DOI: 10.1016/j.celrep.2025.116767

Resource: (Cell Signaling Technology Cat# 2230, RRID:AB_823558)

Curator: @scibot

SciCrunch record: RRID:AB_823558

What is this?

DOI: 10.1016/j.celrep.2025.116767

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2542688

What is this?

DOI: 10.1016/j.celrep.2025.116767

Resource: (Abcam Cat# ab7800, RRID:AB_306091)

Curator: @scibot

SciCrunch record: RRID:AB_306091

What is this?

DOI: 10.1016/j.celrep.2025.116767

Resource: (Proteintech Cat# 13584-1-AP, RRID:AB_2218915)

Curator: @scibot

SciCrunch record: RRID:AB_2218915

What is this?

DOI: 10.1016/j.celrep.2025.116767

Resource: (Abcam Cat# ab21027, RRID:AB_1951138)

Curator: @scibot

SciCrunch record: RRID:AB_1951138

What is this?

DOI: 10.1016/j.celrep.2025.116767

Resource: (Millipore Cat# AB5694, RRID:AB_11212646)

Curator: @scibot

SciCrunch record: RRID:AB_11212646

What is this?

DOI: 10.1016/j.celrep.2025.116766

Resource: (AgriSera Cat# AS07 213, RRID:AB_1031583)

Curator: @scibot

SciCrunch record: RRID:AB_1031583

What is this?

DOI: 10.1016/j.celrep.2025.116766

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_607488

What is this?

DOI: 10.1016/j.celrep.2025.116766

Resource: (MBL International Cat# M185-3L, RRID:AB_11123930)

Curator: @scibot

SciCrunch record: RRID:AB_11123930

What is this?

DOI: 10.1016/j.celrep.2025.116766

Resource: (Easybio Cat# BE0106, RRID:AB_3661754)

Curator: @scibot

SciCrunch record: RRID:AB_3661754

What is this?

DOI: 10.1016/j.celrep.2025.116766

Resource: (Easybio Cat# BE0105, RRID:AB_3661753)

Curator: @scibot

SciCrunch record: RRID:AB_3661753

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (Proteintech Cat# 66192-1-Ig, RRID:AB_2811205)

Curator: @scibot

SciCrunch record: RRID:AB_2811205

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (Proteintech Cat# 66009-1-Ig, RRID:AB_2687938)

Curator: @scibot

SciCrunch record: RRID:AB_2687938

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (Cell Signaling Technology Cat# 83506, RRID:AB_2800018)

Curator: @scibot

SciCrunch record: RRID:AB_2800018

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (Proteintech Cat# SA00001-2, RRID:AB_2722564)

Curator: @scibot

SciCrunch record: RRID:AB_2722564

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (Cell Signaling Technology Cat# 3371, RRID:AB_2140424)

Curator: @scibot

SciCrunch record: RRID:AB_2140424

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (Proteintech Cat# 80031-1-RR, RRID:AB_2918858)

Curator: @scibot

SciCrunch record: RRID:AB_2918858

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (Beyotime Cat# A0216, RRID:AB_2860575)

Curator: @scibot

SciCrunch record: RRID:AB_2860575

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (Cell Signaling Technology Cat# 2535, RRID:AB_331250)

Curator: @scibot

SciCrunch record: RRID:AB_331250

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (Abgent Cat# AP1303a, RRID:AB_2259057)

Curator: @scibot

SciCrunch record: RRID:AB_2259057

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (Bio X Cell Cat# BE0015-1, RRID:AB_1107624)

Curator: @scibot

SciCrunch record: RRID:AB_1107624

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (Cell Signaling Technology Cat# 4410, RRID:AB_1904023)

Curator: @scibot

SciCrunch record: RRID:AB_1904023

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (BioLegend Cat# 100371, RRID:AB_2800555)

Curator: @scibot

SciCrunch record: RRID:AB_2800555

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (BioLegend Cat# 406403, RRID:AB_893531)

Curator: @scibot

SciCrunch record: RRID:AB_893531

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (Cell Signaling Technology Cat# 5831, RRID:AB_10622186)

Curator: @scibot

SciCrunch record: RRID:AB_10622186

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (BioLegend Cat# 100206, RRID:AB_312663)

Curator: @scibot

SciCrunch record: RRID:AB_312663

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (Thermo Fisher Scientific Cat# 12-5965-82, RRID:AB_1257209)

Curator: @scibot

SciCrunch record: RRID:AB_1257209

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (BioLegend Cat# 109219, RRID:AB_893626)

Curator: @scibot

SciCrunch record: RRID:AB_893626

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (BioLegend Cat# 104511, RRID:AB_493565)

Curator: @scibot

SciCrunch record: RRID:AB_493565

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (BioLegend Cat# 100734, RRID:AB_2075238)

Curator: @scibot

SciCrunch record: RRID:AB_2075238

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (BioLegend Cat# 104506, RRID:AB_313109)

Curator: @scibot

SciCrunch record: RRID:AB_313109

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (BioLegend Cat# 104428, RRID:AB_830799)

Curator: @scibot

SciCrunch record: RRID:AB_830799

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (BioLegend Cat# 103012, RRID:AB_312963)

Curator: @scibot

SciCrunch record: RRID:AB_312963

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (BioLegend Cat# 100531, RRID:AB_493374)

Curator: @scibot

SciCrunch record: RRID:AB_493374

What is this?

DOI: 10.1016/j.celrep.2025.116762

Resource: (BioLegend Cat# 100405, RRID:AB_312690)

Curator: @scibot

SciCrunch record: RRID:AB_312690

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: RRID:MGI:3028467

Curator: @scibot

SciCrunch record: RRID:MGI:3028467

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 100728, RRID:AB_493426)

Curator: @scibot

SciCrunch record: RRID:AB_493426

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 116203, RRID:AB_313704)

Curator: @scibot

SciCrunch record: RRID:AB_313704

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 108404, RRID:AB_313369)

Curator: @scibot

SciCrunch record: RRID:AB_313369

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BD Biosciences Cat# 561419, RRID:AB_10611863)

Curator: @scibot

SciCrunch record: RRID:AB_10611863

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 100304, RRID:AB_312669)

Curator: @scibot

SciCrunch record: RRID:AB_312669

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 109121, RRID:AB_2687080)

Curator: @scibot

SciCrunch record: RRID:AB_2687080

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 100555, RRID:AB_2562529)

Curator: @scibot

SciCrunch record: RRID:AB_2562529

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 119706, RRID:AB_2561656)

Curator: @scibot

SciCrunch record: RRID:AB_2561656

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 115530, RRID:AB_830707)

Curator: @scibot

SciCrunch record: RRID:AB_830707

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 104423, RRID:AB_493381)

Curator: @scibot

SciCrunch record: RRID:AB_493381

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 100729, RRID:AB_493702)

Curator: @scibot

SciCrunch record: RRID:AB_493702

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 105838, RRID:AB_2616739)

Curator: @scibot

SciCrunch record: RRID:AB_2616739

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 115504, RRID:AB_313639)

Curator: @scibot

SciCrunch record: RRID:AB_313639

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 506346, RRID:AB_2565955)

Curator: @scibot

SciCrunch record: RRID:AB_2565955

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 105807, RRID:AB_313216)

Curator: @scibot

SciCrunch record: RRID:AB_313216

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 505835, RRID:AB_11219588)

Curator: @scibot

SciCrunch record: RRID:AB_11219588

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 115528, RRID:AB_493735)

Curator: @scibot

SciCrunch record: RRID:AB_493735

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 109104, RRID:AB_313421)

Curator: @scibot

SciCrunch record: RRID:AB_313421

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 108408, RRID:AB_313373)

Curator: @scibot

SciCrunch record: RRID:AB_313373

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 108112, RRID:AB_313349)

Curator: @scibot

SciCrunch record: RRID:AB_313349

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 100750, RRID:AB_2562610)

Curator: @scibot

SciCrunch record: RRID:AB_2562610

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (Bio X Cell Cat# BE0090, RRID:AB_1107780)

Curator: @scibot

SciCrunch record: RRID:AB_1107780

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (Bio X Cell Cat# BE0061, RRID:AB_1125541)

Curator: @scibot

SciCrunch record: RRID:AB_1125541

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 103028, RRID:AB_830785)

Curator: @scibot

SciCrunch record: RRID:AB_830785

What is this?

DOI: 10.1016/j.celrep.2025.116753

Resource: (BioLegend Cat# 149020, RRID:AB_2565703)

Curator: @scibot

SciCrunch record: RRID:AB_2565703

What is this?

DOI: 10.1016/j.celrep.2025.116717

Resource: GraphPad Prism (RRID:SCR_002798)

Curator: @scibot

SciCrunch record: RRID:SCR_002798

What is this?

DOI: 10.1016/j.celrep.2025.116717

Resource: ImageJ (RRID:SCR_003070)

Curator: @scibot

SciCrunch record: RRID:SCR_003070

What is this?

DOI: 10.1016/j.celrep.2025.116717

Resource: Advanced 3D Visualization and Volume Modeling (RRID:SCR_007353)

Curator: @scibot

SciCrunch record: RRID:SCR_007353

What is this?

DOI: 10.1016/j.celrep.2025.116717

Resource: (Jackson ImmunoResearch Labs Cat# 711-545-152, RRID:AB_2313584)

Curator: @scibot

SciCrunch record: RRID:AB_2313584

What is this?

DOI: 10.1016/j.celrep.2025.116717

Resource: (Synaptic Systems Cat# 226 008, RRID:AB_2891278)

Curator: @scibot

SciCrunch record: RRID:AB_2891278

What is this?

DOI: 10.1016/j.celrep.2025.116717

Resource: (Takara Bio Cat# 632496, RRID:AB_10013483)

Curator: @scibot

SciCrunch record: RRID:AB_10013483

What is this?

DOI: 10.1016/j.celrep.2025.116717

Resource: (Molecular Probes Cat# A-11122, RRID:AB_221569)

Curator: @scibot

SciCrunch record: RRID:AB_221569

What is this?

DOI: 10.1016/j.celrep.2025.116717

Resource: (Jackson ImmunoResearch Labs Cat# 711-165-152, RRID:AB_2307443)

Curator: @scibot

SciCrunch record: RRID:AB_2307443

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (Cell Signaling Technology Cat# 13522, RRID:AB_2665370)

Curator: @scibot

SciCrunch record: RRID:AB_2665370

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2809850

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (Thermo Fisher Scientific Cat# MA5-35382, RRID:AB_2849283)

Curator: @scibot

SciCrunch record: RRID:AB_2849283

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (Thermo Fisher Scientific Cat# PA5-35365, RRID:AB_2552675)

Curator: @scibot

SciCrunch record: RRID:AB_2552675

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2807991

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2610404

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2747081

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2854226

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2897854

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2551745

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2636757

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2852221

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (Abcam Cat# ab203859, RRID:AB_2876368)

Curator: @scibot

SciCrunch record: RRID:AB_2876368

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2924681

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (Abcam Cat# ab129076, RRID:AB_11156290)

Curator: @scibot

SciCrunch record: RRID:AB_11156290

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (Abcam Cat# ab124465, RRID:AB_10975633)

Curator: @scibot

SciCrunch record: RRID:AB_10975633

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (Abcam Cat# ab8226, RRID:AB_306371)

Curator: @scibot

SciCrunch record: RRID:AB_306371

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (Abcam Cat# ab97370, RRID:AB_10680261)

Curator: @scibot

SciCrunch record: RRID:AB_10680261

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (Thermo Fisher Scientific Cat# PA5-68240, RRID:AB_2691666)

Curator: @scibot

SciCrunch record: RRID:AB_2691666

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2635913

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (Abcam Cat# ab32042, RRID:AB_725947)

Curator: @scibot

SciCrunch record: RRID:AB_725947

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (Abcam Cat# ab184787, RRID:AB_2827742)

Curator: @scibot

SciCrunch record: RRID:AB_2827742

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (Abcam Cat# ab215206, RRID:AB_2860568)

Curator: @scibot

SciCrunch record: RRID:AB_2860568

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_3164036

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (Abcam Cat# ab245118, RRID:AB_3068617)

Curator: @scibot

SciCrunch record: RRID:AB_3068617

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (Abcam Cat# ab214430, RRID:AB_2938798)

Curator: @scibot

SciCrunch record: RRID:AB_2938798

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (BioLegend Cat# 317414, RRID:AB_571959)

Curator: @scibot

SciCrunch record: RRID:AB_571959

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (BioLegend Cat# 320114, RRID:AB_439754)

Curator: @scibot

SciCrunch record: RRID:AB_439754

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (BioLegend Cat# 100741, RRID:AB_11124344)

Curator: @scibot

SciCrunch record: RRID:AB_11124344

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (BioLegend Cat# 302614, RRID:AB_314284)

Curator: @scibot

SciCrunch record: RRID:AB_314284

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (BioLegend Cat# 126406, RRID:AB_1089113)

Curator: @scibot

SciCrunch record: RRID:AB_1089113

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (BioLegend Cat# 102007, RRID:AB_312856)

Curator: @scibot

SciCrunch record: RRID:AB_312856

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: (BioLegend Cat# 106312, RRID:AB_2563063)

Curator: @scibot

SciCrunch record: RRID:AB_2563063

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:AB_394284

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:AB_2871089

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:AB_2741012

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:AB_2738426

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:AB_2642859

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:AB_2616795

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:AB_2564383

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:AB_2563443

What is this?

DOI: 10.1016/j.cell.2025.11.034

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:AB_2562872

What is this?

DOI: 10.1016/j.bone.2025.117770

Resource: (Cell Signaling Technology Cat# 5536, RRID:AB_10691552)

Curator: @scibot

SciCrunch record: RRID:AB_10691552

What is this?

DOI: 10.1016/j.bone.2025.117770

Resource: (GeneTex Cat# GTX100118, RRID:AB_1080976)

Curator: @scibot

SciCrunch record: RRID:AB_1080976

What is this?

DOI: 10.1016/j.bone.2025.117770

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_11157543

What is this?

DOI: 10.1016/j.bone.2025.117770

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2798685

What is this?

DOI: 10.1016/j.bone.2025.117770

Resource: (Cell Signaling Technology Cat# 8599, RRID:AB_2630347)

Curator: @scibot

SciCrunch record: RRID:AB_2630347

What is this?

DOI: 10.1016/j.bone.2025.117770

Resource: (Cell Signaling Technology Cat# 4228, RRID:AB_659940)

Curator: @scibot

SciCrunch record: RRID:AB_659940

What is this?

DOI: 10.1016/j.bone.2025.117770

Resource: (Cell Signaling Technology Cat# 12556, RRID:AB_2732805)

Curator: @scibot

SciCrunch record: RRID:AB_2732805

What is this?

DOI: 10.1016/j.bone.2025.117770

Resource: (Abcam Cat# ab109012, RRID:AB_2810880)

Curator: @scibot

SciCrunch record: RRID:AB_2810880

What is this?

DOI: 10.1016/j.bone.2025.117770

Resource: (Cell Signaling Technology Cat# 12741, RRID:AB_2617131)

Curator: @scibot

SciCrunch record: RRID:AB_2617131

What is this?

DOI: 10.1016/j.bone.2025.117770

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_368556

What is this?

DOI: 10.1016/j.bone.2025.117770

Resource: (Cell Signaling Technology Cat# 4249, RRID:AB_2165248)

Curator: @scibot

SciCrunch record: RRID:AB_2165248

What is this?

DOI: 10.1016/j.neuroscience.2025.12.033

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:013044

What is this?

DOI: 10.1016/j.isci.2025.114531

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:012897

What is this?

DOI: 10.1016/j.cub.2025.11.074

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:AB_3718094

What is this?

DOI: 10.1016/j.celrep.2025.116797

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:003831

What is this?

https://en.wikipedia.org/wiki/Macaronic_language

It is poetic and incredibly political

The fact that a girl from Nazareth, the bottom of the hierarchy is chosen to birth God says a lot about who God is, someone in touch with the marginalized and lowest of the low

OKVND - mot san choi da tao nen con sot trong cong dong cuoc thu nho vao chat luong dinh cao. Day chac chan se la diem den ma moi bet thu khong the bo lo.

Dia chi: 288 Nam Ky Khoi Nghia, Phuong Vo Thi Sau, Quan 3, Ho Chi Minh, Viet Nam

Email: camdendi18884@gmail.com

Website: https://okvnd.mom/

Dien thoai: (+84) 395942809

okvnd #okvndmom #nhacaiokvnd #trangchuokvnd #appokvnd #songbacokvnd

Social Links:

https://okvnd.mom/

https://okvnd.mom/tai-app-okvnd/

https://okvnd.mom/dang-ky-okvnd/

https://okvnd.mom/nap-tien-okvnd/

https://okvnd.mom/rut-tien-okvnd/

https://okvnd.mom/khuyen-mai-okvnd/

https://okvnd.mom/chinh-sach-bao-mat-okvnd/

https://okvnd.mom/gioi-thieu-okvnd/

https://okvnd.mom/lien-he-okvnd/

https://okvnd.mom/dieu-khoan-dich-vu/

https://okvnd.mom/game-bai-okvnd/

https://okvnd.mom/ceo-ngoc-lan/

https://okvnd.mom/ban-ca-okvnd/

https://okvnd.mom/casino-okvnd/

https://okvnd.mom/the-thao-okvnd/

https://okvnd.mom/xo-so-okvnd/

https://www.facebook.com/okvndmom/

https://www.youtube.com/channel/UCEmhW6tX3OttGPa0QLLeSrA

https://twitter.com/okvndmom

https://www.reddit.com/user/okvndmom/

https://www.pinterest.com/okvndmom/

https://ameblo.jp/okvndmom/entry-12916195106.html

https://gravatar.com/okvndmom

https://www.band.us/band/99261757/intro

https://www.blogger.com/profile/03059450092991625163

https://camdendi18884.wixsite.com/okvndmom

https://www.tumblr.com/okvndmom

https://okvndmom.wordpress.com/

https://www.twitch.tv/okvndmom/about

https://sites.google.com/view/okvndmom/home

https://okvndmom.webflow.io/

https://bookmarksclub.com/backlink/okvndmom/

https://okvndmom.mystrikingly.com/

https://okvndmom.amebaownd.com/

https://telegra.ph/okvndmom-07-13

https://mk.gta5-mods.com/users/okvndmom

https://68732e5d9b667.site123.me/

https://myspace.com/okvndmom

https://scholar.google.com/citations?hl=vi&user=sv5P2_kAAAAJ

https://www.pearltrees.com/okvndmom/item726517039

https://okvndmom.localinfo.jp/

https://okvndmom.shopinfo.jp/

https://okvndmom.hashnode.dev/okvndmom

https://okvndmom.themedia.jp/

https://rapidapi.com/user/okvndmom

https://730802.8b.io/

https://okvndmom.theblog.me/

https://fliphtml5.com/homepage/vwdfz/okvndmom/

https://okvndmom.therestaurant.jp/

https://ask.mallaky.com/?qa=user/okvndmom

https://okvndmom.website3.me/

https://www.quora.com/profile/Okvndmom

https://okvndmom.pixieset.com/

https://okvndmom.gumroad.com/

https://flipboard.com/@okvndmom/okvndmom-guvuh747y

https://www.threadless.com/@okvndmom/activity

https://wakelet.com/@okvndmom

https://www.magcloud.com/user/okvndmom

https://hackmd.io/@okvndmom/okvndmom

https://okvndmom.blogspot.com/2025/07/okvndmom.html

https://okvndmom.doorkeeper.jp/

https://okvndmom.storeinfo.jp/

https://velog.io/@okvndmom/about

https://bato.to/u/2818889-okvndmom

https://zb3.org/okvndmom/okvndmom

https://github.com/okvndmom

https://community.fabric.microsoft.com/t5/user/viewprofilepage/user-id/1308289

https://bit.ly/4kEgBpv

https://tinyurl.com/okvndmom

https://tawk.to/okvndmom

https://gitlab.com/okvndmom

https://rebrand.ly/okvndmom

https://www.question-ksa.com/user/okvndmom

https://bulkwp.com/support-forums/users/okvndmom/

https://orcid.org/0009-0006-0979-7660

https://community.cisco.com/t5/user/viewprofilepage/user-id/1897209

https://linktr.ee/okvndmom

https://archive.org/details/@okvndmom/web-archive

https://wpfr.net/support/utilisateurs/okvndmom

https://ameblo.jp/okvndmom

https://plaza.rakuten.co.jp/okvndmom/diary/202507130000/

https://pad.darmstadt.social/s/GVjTnHJ73

https://pixabay.com/users/51296485/

https://disqus.com/by/okvndmom/about/

https://www.reverbnation.com/artist/okvndmom

https://es.gta5-mods.com/users/okvndmom

https://www.gamblingtherapy.org/forum/users/okvndmom/

https://heylink.me/okvndmom/

https://forum.m5stack.com/user/okvndmom/

https://app.readthedocs.org/profiles/okvndmom/

https://gitee.com/okvndmom

https://public.tableau.com/app/profile/okvnd.mom/viz/okvndmom/Sheet1#1

https://connect.garmin.com/modern/profile/b5f0bfed-db89-462b-8a52-1ce3554f4cd2

https://www.pixiv.net/en/users/117941688

https://community.amd.com/t5/user/viewprofilepage/user-id/513598

https://readtoto.com/u/2818889-okvndmom

https://s.id/0i3cj

https://qna.habr.com/user/okvndmom

https://linkr.bio/okvndmom

https://www.bark.com/en/gb/company/okvndmom/MLaL4B/

https://pastebin.com/u/okvndmom

https://www.storeboard.com/okvndmom

https://etextpad.com/84zl4vokeu

https://md.darmstadt.ccc.de/s/1bgmCSBfX

https://vc.ru/id5099760

https://qiita.com/okvndmom

https://comicvine.gamespot.com/profile/okvndmom/

https://padlet.com/okvndmom/okvndmom

https://3dwarehouse.sketchup.com/by/okvndmom

https://muckrack.com/okvnd-mom/bio

https://hedgedoc.k8s.eonerc.rwth-aachen.de/s/oOso1KxPR

https://connect.informs.org/network/speakerdirectory/speaker?UserKey=20c22721-fc67-4442-bc5c-0198025479cd

https://nl.gta5-mods.com/users/okvndmom

https://openlibrary.org/people/okvndmom

https://anyflip.com/homepage/avazj#About

https://lu.ma/user/okvndmom

Tôi chưa hiểu lắm

V

V

False???

配列について

配列とは同じ型の変数を一気に作る方法でありかっこの中の <br /> [数字]の分だけ作れる

int score [4]はint型のscoreという名前の変数を４つ作成する　　ことになる

↓配列の必要性と宣言の仕方↓

参考授業資料(第7回)5ページ

関数について

同じ処理を複数書かないように，処理をまとまりにしたものを関数という

プログラムを綺麗にかけたり，処理変更の時に間違えを減らせる

参考授業資料(第4回)8ページ

配列と関数について

関数に配列を返す時，return文では配列全体を返すことはできないことに注意

参考授業資料(第9回)20ページ

プロトタイプ宣言について

プログラムはmain関数から読み込んでいくため，先に関数を作っていると流れが見えにくい

そのため，プロトタイプ宣言をしてmain関数を先に書いておこう

参考授業資料(第4回)17ページ

二次元配列と二重ループについて

二次元配列を表示，格納する時は二重ループを使う

処理の流れをフローチャートで覚えると分かりやすい

↓参考図↓

参考授業資料(第9回)22ページ

関数について

同じ処理を複数書かないように，処理をまとまりにしたものを関数という

プログラムを綺麗にかけたり，処理変更の時に間違えを減らせる

参考授業資料(第4回)8ページ

関数の引数と戻り値について

戻り値がない場合にはvoid型を使う

参考授業資料(第4回)12ページ

二次元配列について

多次元配列はまずは図で覚えると分かりやすい

図は講義資料を参考にしよう

参考授業資料(第9回)16ページ

プロトタイプ宣言について

プログラムはmain関数から読み込んでいくため，先に関数を作っていると流れが見えにくい

そのため，プロトタイプ宣言をしてmain関数を先に書いておこう

参考授業資料(第4回)17ページ

構造体について

関連するデータを1つのまとまりとして持つと覚えたらわかりやすい

構造体の構成について講義資料を参考に覚えよう

参考授業資料(第10回)6ページ

構造体と関数について

構造体は関数に引数としても戻り値としても渡せる

参考授業資料(第11回)30ページ

構造体について

int data[N][2]とchar id_num[N][M]を構造体を使って1つにまとめている

使う時はdatas[O].data[O] or id_num[O]とかく

参考授業資料(第11回)6ページ

プロトタイプ宣言について

プログラムはmain関数から読み込んでいくため，先に関数を作っていると流れが見えにくい

そのため，プロトタイプ宣言をしてmain関数を先に書いておこう

参考授業資料(第4回)17ページ

typedef宣言について

構造体はそのままだと名前が長くて間違えやすい

例）

struct data { ...で書いてあったら

main関数で使う時は　struct data xxx;と宣言する必要がある

typedef struct {...}DATA;で書いてあったら

main関数で使う時は　DATA xxx;と宣言するだけ

講義資料も参考にtypedef宣言について知ろう

参考授業資料(第11回)30ページ

関数の引数と戻り値について

戻り値がない場合にはvoid型を使う

参考授業資料(第4回)12ページ

関数について

同じ処理を複数書かないように，処理をまとまりにしたものを関数という

プログラムを綺麗にかけたり，処理変更の時に間違えを減らせる

参考授業資料(第4回)8ページ

プロトタイプ宣言について

プログラムはmain関数から読み込んでいくため，先に関数を作っていると流れが見えにくい

そのため，プロトタイプ宣言をしてmain関数を先に書いておこう

参考授業資料(第4回)17ページ

プロトタイプ宣言について

プログラムはmain関数から読み込んでいくため，先に関数を作っていると流れが見えにくい

そのため，プロトタイプ宣言をしてmain関数を先に書いておこう

参考授業資料(第4回)17ページ

配列について

参考授業資料(第9回)4ページ

配列と関数について

関数に配列を返す時，return文では配列全体を返すことはできないことに注意

参考授業資料(第9回)20ページ

関数について

同じ処理を複数書かないように，処理をまとまりにしたものを関数という

プログラムを綺麗にかけたり，処理変更の時に間違えを減らせる

参考授業資料(第4回)8ページ

#defineについて

#define文を使うことによって配列をたくさん使うプログラムをかく時に間違いを減らすことが出来る

[参考授業資料(第7回)30ページ](http://kadai.cse.ce.nihon-u.ac.jp/image/pdf/P基礎_No7_allNoSoundNoAnime.pdf#page=30

構造体変数の宣言について

参考授業資料(第10回)9ページ

構造体について

関連するデータを1つのまとまりとして持つと覚えたらわかりやすい

構造体の構成について講義資料を参考に覚えよう

参考授業資料(第10回)6ページ

typedef宣言について

構造体はそのままだと名前が長くて間違えやすい

例）

struct data { ...で書いてあったら

main関数で使う時は　struct data xxx;と宣言する必要がある

typedef struct {...}DATA;で書いてあったら

main関数で使う時は　DATA xxx;と宣言するだけ

講義資料も参考にtypedef宣言について知ろう

参考授業資料(第11回)30ページ

構造体と関数について

構造体は関数に引数としても戻り値としても渡せる

参考授業資料(第11回)30ページ

#defineについて

#define文を使うことによって配列をたくさん使うプログラムをかく時に間違いを減らすことが出来る

[参考授業資料(第7回)30ページ](http://kadai.cse.ce.nihon-u.ac.jp/image/pdf/P基礎_No7_allNoSoundNoAnime.pdf#page=30

構造体について

int data[N][2]とchar id_num[N][M]を構造体を使って1つにまとめている

使う時はdatas[O].data[O] or id_num[O]とかく

参考授業資料(第11回)6ページ

プロトタイプ宣言について

プログラムはmain関数から読み込んでいくため，先に関数を作っていると流れが見えにくい

そのため，プロトタイプ宣言をしてmain関数を先に書いておこう

参考授業資料(第4回)17ページ

typedef宣言について

構造体はそのままだと名前が長くて間違えやすい

例）

struct data { ...で書いてあったら

main関数で使う時は　struct data xxx;と宣言する必要がある

typedef struct {...}DATA;で書いてあったら

main関数で使う時は　DATA xxx;と宣言するだけ

講義資料も参考にtypedef宣言について知ろう

参考授業資料(第11回)30ページ

#defineについて

#define文を使うことによって配列をたくさん使うプログラムをかく時に間違いを減らすことが出来る

参考授業資料(第7回)30ページ

構造体と関数について

構造体は関数に引数としても戻り値としても渡せる

参考授業資料(第11回)30ページ

関数の引数と戻り値について

戻り値がない場合にはvoid型を使う

参考授業資料(第4回)12ページ

関数について

同じ処理を複数書かないように，処理をまとまりにしたものを関数という

プログラムを綺麗にかけたり，処理変更の時に間違えを減らせる

参考授業資料(第4回)8ページ

プロトタイプ宣言について

プログラムはmain関数から読み込んでいくため，先に関数を作っていると流れが見えにくい

そのため，プロトタイプ宣言をしてmain関数を先に書いておこう

参考授業資料(第4回)17ページ

無限ループについて

while(1)は無限ループが行われる.

参考授業資料(第3回)22ページ

break文について

ループ処理において強制終了するためにbreak文が使われる

参考授業資料(第3回)23ページ

※小数点表示に注意 （小数点以下を2桁に指定したい時は%.2fとする）

参考授業資料(第2回)37ページ

無限ループについて

while(1)は無限ループが行われる

参考授業資料(第3回)22ページ

break文について

ループ処理において強制終了するためにbreak文が使われる

参考授業資料(第3回)23ページ

論理演算子について

論理演算子を使ってelse ifの条件を書いていく

例）3または7の倍数を判定するときif，else if文の条件

3と7の倍数の時→if(n % 3 == 0 || n % 7== 0)

参考授業資料(第2回)25ページ

プロトタイプ宣言について

プログラムはmain関数から読み込んでいくため，先に関数を作っていると流れが見えにくい

そのため，プロトタイプ宣言をしてmain関数を先に書いておこう

参考授業資料(第4回)17ページ

構造体について

int data[N][2]とchar id_num[N][M]を構造体を使って1つにまとめている

使う時はdatas[O].data[O] or id_num[O]とかく

参考授業資料(第11回)6ページ

typedef宣言について

構造体はそのままだと名前が長くて間違えやすい

例）

struct data { ...で書いてあったら

main関数で使う時は　struct data xxx;と宣言する必要がある

typedef struct {...}DATA;で書いてあったら

main関数で使う時は　DATA xxx;と宣言するだけ

講義資料も参考にtypedef宣言について知ろう

参考授業資料(第11回)30ページ

#defineについて

#define文を使うことによって配列をたくさん使うプログラムをかく時に間違いを減らすことが出来る

参考授業資料(第7回)30ページ

構造体と関数について

構造体は関数に引数としても戻り値としても渡せる

参考授業資料(第11回)30ページ

文字列の配列について

char型の二次元配列を覚えるにはまずは図で表したら分かりやすい

二次元配列に文字列を入れる方法

6行目の「char a[3][20]」で20文字まで入る配列を3つ用意して、その3つの配列にどんな文字を入れるかの初期化の作業

ここでは「a[0]」の配列に「Nagasawa Masami」の文字を入れている

図は講義資料を参考にしてみよう

参考授業資料(第10回)25ページ

配列について

要素数は0から始まることに注意

参考授業資料(第7回)13ページ

配列と関数について

関数に配列を返す時，return文では配列全体を返すことはできないことに注意

参考授業資料(第9回)20ページ

プロトタイプ宣言について

プログラムはmain関数から読み込んでいくため，先に関数を作っていると流れが見えにくい

そのため，プロトタイプ宣言をしてmain関数を先に書いておこう

参考授業資料(第4回)17ページ

#defineについて

#define文を使うことによって配列をたくさん使うプログラムをかく時に間違いを減らすことが出来る

参考授業資料(第7回)30ページ

※数学ライブラリ関数の#include < math.h > の三角関数「sin(x)」

参考授業資料(第4回)29ページ

数学ライブラリ関数の#include < math.h >について

sinやcosなどを使用したいときに必要なライブラリー

↓数学ライブラリ一覧↓

参考授業資料(第4回)28ページ

※加算と表示を1行にまとめる書き方に注意 参考授業資料(第1回)23ページ

※printfでの「％」の表示方法に注意

参考授業資料(第1回)31ページ

※小数点表示に注意 （小数点以下を2桁に指定したい時は%.2fとする）

参考授業資料(第2回)37ページ

Test

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

Reviewer #1* (Evidence, reproducibility and clarity (Required)):

Summary: In this study, the authors used proximity proteomics in U2OS cells to identify several E3 ubiquitin ligases recruited to stress granules (SGs), and they focused on MKRN2 as a novel regulator. They show that MKRN2 localization to SGs requires active ubiquitination via UBA1. Functional experiments demonstrated that MKRN2 knockdown increases the number of SG condensates, reduces their size, slightly raises SG liquidity during assembly, and slows disassembly after heat shock. Overexpression of MKRN2-GFP combined with confocal imaging revealed co-localization of MKRN2 and ubiquitin in SGs. By perturbing ubiquitination (using a UBA1 inhibitor) and inducing defective ribosomal products (DRiPs) with O-propargyl puromycin, they found that both ubiquitination inhibition and MKRN2 depletion lead to increased accumulation of DRiPs in SGs. The authors conclude that MKRN2 supports granulostasis, the maintenance of SG homeostasis , through its ubiquitin ligase activity, preventing pathological DRiP accumulation within SGs.

Major comments: - Are the key conclusions convincing? The key conclusions are partially convincing. The data supporting the role of ubiquitination and MKRN2 in regulating SG condensate dynamics are coherent, well controlled, and consistent with previous literature, making this part of the study solid and credible. However, the conclusions regarding the ubiquitin-dependent recruitment of MKRN2 to SGs, its relationship with UBA1 activity, the functional impact of the MKRN2 knockdown for DRiP accumulation are less thoroughly supported. These aspects would benefit from additional mechanistic evidence, validation in complementary model systems, or the use of alternative methodological approaches to strengthen the causal connections drawn by the authors. - Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? The authors should qualify some of their claims as preliminary. 1) MKRN2 recruitment to SGs (ubiquitin-dependent): The proteomics and IF data are a reasonable starting point, but they do not yet establish that MKRN2 is recruited from its physiological localization to SGs in a ubiquitin-dependent manner. To avoid overstating this point the authors should qualify the claim and/or provide additional controls: show baseline localization of endogenous MKRN2 under non-stress conditions (which is reported in literature to be nuclear and cytoplasmatic), include quantification of nuclear/cytoplasmic distribution, and demonstrate a shift into bona fide SG compartments after heat shock. Moreover, co-localization of overexpressed GFP-MKRN2 with poly-Ub (FK2) should be compared to a non-stress control and to UBA1-inhibition conditions to support claims of stress- and ubiquitination-dependent recruitment. *

Authors: We will stain cells for endogenous MKRN2 and quantify nuc/cyto ratio of MKRN2 without heat stress, without heat stress + TAK243, with HS and with HS + TAK243. We will do the same in the MKRN2-GFP overexpressing line while also staining for FK2.

*2) Use and interpretation of UBA1 inhibition: UBA1 inhibition effectively blocks ubiquitination globally, but it is non-selective. The manuscript should explicitly acknowledge this limitation when interpreting results from both proteomics and functional assays. Proteomics hits identified under UBA1 inhibition should be discussed as UBA1-dependent associations rather than as evidence for specific E3 ligase recruitment. The authors should consider orthogonal approaches before concluding specificity. *

Authors: We have acknowledged the limitation of using only TAK243 in our study by rephrasing statements about dependency on “ubiquitination” to “UBA1-dependent associations”.

* 3) DRiP accumulation and imaging quality: The evidence presented in Figure 5 is sufficient to substantiate the claim that DRiPs accumulate in SGs upon ubiquitination inhibition or MKRN2 depletion but to show that the event of the SGs localization and their clearance from SGs during stress is promoted by MKRN3 ubiquitin ligase activity more experiments would be needed. *

Authors: We have acknowledged the fact that our experiments do not include DRiP and SG dynamics assays using ligase-dead mutants of MKRN2 by altering our statement regarding MKRN2-mediated ubiquitination of DRiPs in the text (as proposed by reviewer 1).

*- Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. Yes, a few targeted experiments would strengthen the conclusions without requiring the authors to open new lines of investigation. 1) Baseline localization of MKRN2: It would be important to show the baseline localization of endogenous and over-expressed MKRN2 (nuclear and cytoplasmic) under non-stress conditions and prior to ubiquitination inhibition. This would provide a reference to quantify redistribution into SGs and demonstrate recruitment in response to heat stress or ubiquitination-dependent mechanisms. *

Authors: We thank the reviewer for bringing this important control. We will address it in revisions.

We will quantify the nuclear/cytoplasmic distribution of endogenous and GFP-MKRN2 under control, TAK243, heat shock, and combined conditions, and assess MKRN2–ubiquitin colocalization by FK2 staining in unstressed cells.

* 2) Specificity of MKRN2 ubiquitin ligase activity: to address the non-specific effects of UBA1 inhibition and validate that observed phenotypes depend on MKRN2's ligase activity, the authors could employ a catalytically inactive MKRN2 mutant in rescue experiments. Comparing wild-type and catalytic-dead MKRN2 in the knockdown background would clarify the causal role of MKRN2 activity in SG dynamics and DRiP clearance. *

Authors: We thank the reviewer for this suggestion and have altered the phrasing of some of our statements in the text accordingly.

* 3) Ubiquitination linkage and SG marker levels: While the specific ubiquitin linkage type remains unknown, examining whether MKRN2 knockdown or overexpression affects total levels of key SG marker proteins would be informative. This could be done via Western blotting of SG markers along with ubiquitin staining, to assess whether MKRN2 influences protein stability or turnover through degradative or non-degradative ubiquitination. Such data would strengthen the mechanistic interpretation while remaining within the current study's scope. *

Authors: We thank the reviewer for requesting and will address it by performing MKRN2 KD and perform Western blot for G3BP1.

*

• Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments. The experiments suggested in points 1 and 3 are realistic and should not require substantial additional resources beyond those already used in the study. • Point 1 (baseline localization of MKRN2): This involves adding two control conditions (no stress and no ubiquitination inhibition) for microscopy imaging. The setup is essentially the same as in the current experiments, with time requirements mainly dependent on cell culture growth and imaging. Overall, this could be completed within a few weeks. • Point 3 (SG marker levels and ubiquitination): This entails repeating the existing experiment and adding a Western blot for SG markers and ubiquitin. The lab should already have the necessary antibodies, and the experiment could reasonably be performed within a couple of weeks. • Point 2 (catalytically inactive MKRN2 mutant and rescue experiments): This is likely more time-consuming. Designing an effective catalytic-dead mutant depends on structural knowledge of MKRN2 and may require additional validation to confirm loss of catalytic activity. If this expertise is not already present in the lab, it could significantly extend the timeline. Therefore, this experiment should be considered only if similarly recommended by other reviewers, as it represents a higher resource and time investment.

Overall, points 1 and 3 are highly feasible, while point 2 is more substantial and may require careful planning.

• Are the data and the methods presented in such a way that they can be reproduced? Yes. The methodologies used in this study to analyze SG dynamics and DRiP accumulation are well-established in the field and should be reproducible, particularly by researchers experienced in stress granule biology. Techniques such as SG assembly and disassembly assays, use of G3BP1 markers, and UBA1 inhibition are standard and clearly described. The data are generally presented in a reproducible manner; however, as noted above, some results would benefit from additional controls or complementary experiments to fully support specific conclusions.

• Are the experiments adequately replicated and statistical analysis adequate? Overall, the experiments in the manuscript appear to be adequately replicated, with most assays repeated between three and five times, as indicated in the supplementary materials. The statistical analyses used are appropriate and correctly applied to the datasets presented. However, for Figure 5 the number of experimental replicates is not reported. This should be clarified, and if the experiment was not repeated sufficiently, additional biological replicates should be performed. Given that this figure provides central evidence supporting the conclusion that DRiP accumulation depends on ubiquitination-and partly on MKRN2's ubiquitin ligase activity-adequate replication is essential. *

Authors: We thank the reviewer for noting this accidental omission. We now clarify in the legend of Figure 5 that the experiments with DRiPs were replicated three times.

Minor comments: - Specific experimental issues that are easily addressable. • For the generation and the validation of MKRN2 knockdown in UOS2 cells data are not presented in the results or in the methods sections to demonstrate the effective knockdown of the protein of interest. This point is quite essential to demonstrate the validity of the system used

Authors: We thank the reviewer for requesting and will address it by performing MKRN2 KD and perform Western blot and RT-qPCR.

• * In the supplementary figure 2 it would be useful to mention if the Western Blot represent the input (total cell lysates) before the APEX-pulldown or if it is the APEX-pulldown loaded for WB. There is no consistence in the difference of biotynilation between different replicates shown in the 2 blots. For example in R1 and R2 G3BP1-APX TAK243 the biotynilation is one if the strongest condition while on the left blot, in the same condition comparison samples R3 and R4 are less biotinilated compared to others. It would be useful to provide an explanation for that to avoid any confusion for the readers. * Authors: We have added a mention in the legend of Figure S2 that these are total cell lysates before pulldown. The apparent differences in biotin staining are small and not sufficient to question the results of our APEX-proteomics.

• * In Figure 2D, endogenous MKRN2 localization to SGs appears reduced following UBA1 inhibition. However, it is not clear whether this reduction reflects a true relocalization or a decrease in total MKRN2 protein levels. To support the interpretation that UBA1 inhibition specifically affects MKRN2 recruitment to SGs rather than its overall expression, the authors should provide data showing total MKRN2 levels remain unchanged under UBA1 inhibition, for example via Western blot of total cell lysates. * Authors: Based on first principles in regulation of gene expression, it is unlikely that total MKRN2 expression levels would decrease appreciably through transcriptional or translational regulation within the short timescale of these experiments (1 h TAK243 pretreatment followed by 90 min of heat stress).

• * DRIPs accumulation is followed during assembly but in the introduction is highlighted the fact that ubiquitination events, other reported E3 ligases and in this study data on MKRN2 showed that they play a crucial role in the disassembly of SGs which is also related with cleareance of DRIPs. Authors could add tracking DRIPs accumulation during disassembly to be added to Figure 5. I am not sure about the timeline required for this but I am just adding as optional if could be addressed easily. * Authors: We thank the reviewer for proposing this experimental direction. However, in a previous study (Ganassi et al., 2016; 10.1016/j.molcel.2016.07.021), we demonstrated that DRiP accumulation during the stress granule assembly phase drives conversion to a solid-like state and delays stress granule disassembly. It is therefore critical to assess DRiP enrichment within stress granules immediately after their formation, rather than during the stress recovery phase, as done here.

• * The authors should clarify in the text why the cutoff used for the quantification in Figure 5D (PC > 3) differs from the cutoff used elsewhere in the paper (PC > 1.5). Providing a rationale for this choice will help the reader understand the methodological consistency and ensure that differences in thresholds do not confound interpretation of the results. * Authors: We thank the reviewer for this question. The population of SGs with a DRiP enrichment > 1.5 represents SGs with a significant DRiP enrichment compared to the surrounding (background) signal. As explained in the methods, the intensity of DRiPs inside each SG is corrected by the intensity of DRiPs two pixels outside of each SG. Thus, differences in thresholds between independent experimental conditions (5B versus 5D) do not confound interpretation of the results but depend on overall staining intensity that can different between different experimental conditions. Choosing the cut-off > 3 allows to specifically highlight the population of SGs that are strongly enriched with DRiPs. MKRN2 silencing caused a strong DRiP enrichment in the majority of the SGs analyzed and therefore we chose this way of data representation. Note that the results represent the average of the analysis of 3 independent experiments with high numbers of SGs automatically segmented and analyzed/experiment. Figure 5A, B: n = 3 independent experiments; number of SGs analyzed per experiment: HS + OP-puro (695; 1216; 952); TAK-243 + HS + OP-puro (1852; 2214; 1774). Figure 5C, D: n = 3 independent experiments; number of SGs analyzed per experiment: siRNA control, HS + OP-puro (1984; 1400; 1708); siRNA MKRN2, HS + OP-puro (912; 1074; 1532).

• * For Figure 3G, the authors use over-expressed MKRN2-GFP to assess co-localization with ubiquitin in SGs. Given that a reliable antibody for endogenous MKRN2 is available and that a validated MKRN2 knockdown line exists as an appropriate control, this experiment would gain significantly in robustness and interpretability if co-localization were demonstrated using endogenous MKRN2. In the current over-expression system, MKRN2-GFP is also present in the nucleus, whereas the endogenous protein does not appear nuclear under the conditions shown. This discrepancy raises concerns about potential over-expression artifacts or mislocalization. Demonstrating co-localization using endogenous MKRN2 would avoid confounding effects associated with over-expression. If feasible, this would be a relatively straightforward experiment to implement, as it relies on tools (antibody and knockdown line) already described in the manuscript.

* Authors: We thank the reviewer for requesting and will address it by performing MKRN2 KD, FK2 immunofluorescence microscopy and perform SG partition coefficient analysis.

* - Are prior studies referenced appropriately? • From line 54 to line 67, the manuscript in total cites eight papers regarding the role of ubiquitination in SG disassembly. However, given the use of UBA1 inhibition in the initial MS-APEX experiment and the extensive prior literature on ubiquitination in SG assembly and disassembly under various stress conditions, the manuscript would benefit from citing additional relevant studies to provide more specifc examples. Expanding the references would provide stronger context, better connect the current findings to prior work, and emphasize the significance of the study in relation to established literature *

Authors: We have added citations for the relevant studies.

• *

At line 59, it would be helpful to note that G3BP1 is ubiquitinated by TRIM21 through a Lys63-linked ubiquitin chain. This information provides important mechanistic context, suggesting that ubiquitination of SG proteins in these pathways is likely non-degradative and related to functional regulation of SG dynamics rather than protein turnover. * Authors: The reviewer is correct. We have added to the text that G3BP1 is ubiquitinated through a Lys63-linked ubiquitin chain.

• *

When citing references 16 and 17, which report that the E3 ligases TRIM21 and HECT regulate SG formation, the authors should provide a plausible explanation for why these specific E3 ligases were not detected in their proteomics experiments. Differences could arise from the stress stimulus used, cell type, or experimental conditions. Similarly, since MKRN2 and other E3 ligases identified in this study have not been reported in previous works, discussing these methodological or biological differences would help prevent readers from questioning the credibility of the findings. It would also be valuable to clarify in the Conclusion that different types of stress may activate distinct ubiquitination pathways, highlighting context-dependent regulation of SG assembly and disassembly. * Authors: We thank the reviewer for this suggestion. We added to the discussion plausible explanations for why our study identified new E3 ligases.

• *

Line 59-60: when referring to the HECT family of E3 ligases involved in ubiquitination and SG disassembly, it would be more precise to report the specific E3 ligase identified in the cited studies rather than only the class of ligase. This would provide clearer mechanistic context and improve accuracy for readers. * Authors: We have added this detail to the discussion.

• *

The specific statement on line 182 "SG E3 ligases that depend on UBA1 activity are RBULs" should be supported by reference. * Authors: We have added citations to back up our claim that ZNF598, CNOT4, MKRN2, TRIM25 and TRIM26 exhibit RNA-binding activity.

*- Are the text and figures clear and accurate?

• In Supplementary Figure 1, DMSO is shown in green and the treatment in red, whereas in the main figures (Figure 1B and 1F) the colours in the legend are inverted. To avoid confusion, the colour coding in figure legends should be consistent across all figures throughout the manuscript. *

Authors: We have made the colors consistent across the main and supplementary figures.

• *

At line 79, the manuscript states that "inhibition of ubiquitination delayed fluorescence recovery dynamics of G3BP1-mCherry, relative to HS-treated cells (Figure 1F, Supplementary Fig. 6A)." However, the data shown in Figure 1F appear to indicate the opposite effect: the TAK243-treated condition (green curve) shows a faster fluorescence recovery compared to the control (red curve). This discrepancy between the text and the figure should be corrected or clarified, as it may affect the interpretation of the role of ubiquitination in SG dynamics. * Authors: Good catch. We now fixed the graphical mistake (Figure 1F and S6).

• * Line 86: adjust a missing bracket * Authors: Thank you, we fixed it.

• *

There appears to be an error in the legend of Supplementary Figure 3: the legend states that the red condition (MKRN2) forms larger aggregates, but both the main Figure 3C of the confocal images and the text indicate that MKRN2 (red) forms smaller aggregates. Please correct the legend and any corresponding labels so they are consistent with the main figure and the text. The authors should also double-check that the figure panel order, color coding, and statistical annotations match the legend and the descriptions in the Results section to avoid reader confusion.

* Authors: This unfortunate graphical mistake has been corrected.

• * At lines 129-130, the manuscript states that "FRAP analysis demonstrated that MKRN2 KD resulted in a slight increase in SG liquidity (Fig. 3F, Supplementary Fig. 6B)." However, the data shown in Figure 3F appear to indicate the opposite trend: the MKRN2 KD condition (red curve) exhibits a faster fluorescence recovery compared to the control (green curve). This discrepancy between the text and the figure should be corrected or clarified, as it directly affects the interpretation of MKRN2's role in SG disassembly. Ensuring consistency between the written description and the plotted FRAP data is essential for accurate interpretation. * Authors: We thank the reviewer and clarify in the legend of Figure 3F and the Results the correct labels: indeed faster fluorescence recovery seen in MKRN2 KD is correctly interpreted as increased liquidity in the text.

• *

At lines 132-133, the manuscript states: "Then, to further test the impact of MKRN2 on SG dynamics, we overexpressed MKRN2-GFP and observed that it was recruited to SG (Fig. 3G)." This description should be corrected or clarified, as the over-expressed MKRN2-GFP also appears to localize to the nucleus. * Authors: The text has been modified to reflect both the study of MKRN2 localization to SGs and of nuclear localization.

• *

At lines 134-135, the manuscript states that the FK2 antibody detects "free ubiquitin." This is incorrect. FK2 does not detect free ubiquitin; it recognizes only ubiquitin conjugates, including mono-ubiquitinated and poly-ubiquitinated proteins. The text should be corrected accordingly to avoid misinterpretation of the immunostaining data. * Authors: Thank you for pointing out this error. We have corrected it.

• * Figure 5A suffers from poor resolution, and no scale bar is provided, which limits interpretability. Additionally, the ROI selected for the green channel (DRIPs) appears to capture unspecific background staining, while the most obvious DRIP spots are localized in the nucleus. The authors should clarify this in the text, improve the image quality if possible, and ensure that the ROI accurately represents DRIP accumulation - in SGs rather than background signal. * Authors: We thank the reviewer for pointing the sub-optimal presentation of this figure. We modified Figure 5A to improve image quality and interpretation. Concerning the comment that “the most obvious DRIP spots are localized in the nucleus”, this is in line with our previous findings demonstrating that a fraction of DRiPs accumulates in nucleoli (Mediani et al. 2019 10.15252/embj.2018101341). To avoid misinterpretation, we modified Figure 5A as follows: (i) we provide a different image for control cells, exposed to heat shock and OP-puro; (ii) we select a ROI that only shows a few stress granules; (iii) we added arrowheads to indicate the nucleoli that are strongly enriched for DRiPs; (iv) we include a dotted line to show the nuclear membrane, helping to distinguish cytoplasm and nucleus in the red and green channel. We also include the scale bars (5 µm) in the image.

* Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

• In the first paragraph following the APEX proteomics results, the authors present validation data exclusively for MKRN2, justifying this early focus by stating that MKRN2 is the most SG-depleted E3 ligase. However, in the subsequent paragraph they introduce the RBULs and present knockdown data for MKRN2 along with two additional E3 ligases identified in the screen, before once again emphasizing that MKRN2 is the most SG-depleted ligase and therefore the main focus of the study. For clarity and logical flow, the manuscript would benefit from reordering the narrative. Specifically, the authors should first present the validation data for all three selected E3 ligases, and only then justify the decision to focus on MKRN2 for in-depth characterization. In addition to the extent of its SG depletion, the authors may also consider providing biologically relevant reasons for prioritizing MKRN2 (e.g., domain architecture, known roles in stress responses, or prior evidence of ubiquitination-related functions). Reorganizing this section would improve readability and better guide the reader through the rationale for the study's focus.*

Authors: We thank the reviewer for this suggested improvement to our “storyline”. As suggested by the reviewer, we have moved the IF validation of MKRN2 to the following paragraph in order to improve the flow of the manuscript. We added additional justification to prioritizing MKRN2 citing (Youn et al. 2018 and Markmiller et al. 2018).

• *

At lines 137-138, the manuscript states: "Together these data indicate that MKRN2 regulates the assembly dynamics of SGs by promoting their coalescence during HS and can increase SG ubiquitin content." While Figure 3G shows some co-localization of MKRN2 with ubiquitin, immunofluorescence alone is insufficient to claim an increase in SG ubiquitin content. This conclusion should be supported by orthogonal experiments, such as Western blotting, in vitro ubiquitination assays, or immunoprecipitation of SG components. Including a control under no-stress conditions would also help demonstrate that ubiquitination increases specifically in response to stress. The second part of the statement should therefore be rephrased to avoid overinterpretation, for example:"...and may be associated with increased ubiquitination within SGs, as suggested by co-localization, pending further validation by complementary assays." * Authors: The statement has been rephrased in a softer way as suggested by the reviewer.

• At line 157, the statement: "Therefore, we conclude that MKRN2 ubiquitinates a subset of DRiPs, avoiding their accumulation inside SGs" should be rephrased as a preliminary observation. While the data support a role for MKRN2 in SG disassembly and a reduction of DRIPs, direct ubiquitination of DRIPs by MKRN2 has not been demonstrated. A more cautious phrasing would better reflect the current evidence and avoid overinterpretation. * * *Authors: We thank the reviewer for this suggestion and have altered the phrasing of this statement accordingly.

*Reviewer #1 (Significance (Required)):

General assessment: provide a summary of the strengths and limitations of the study. What are the strongest and most important aspects? What aspects of the study should be improved or could be developed?

• This study provides a valuable advancement in understanding the role of ubiquitination in stress granule (SG) dynamics and the clearance of SGs formed under heat stress. A major strength is the demonstration of how E3 ligases identified through proteomic screening, particularly MKRN2, influence SG assembly and disassembly in a ubiquitination- and heat stress-dependent manner. The combination of proteomics, imaging, and functional assays provides a coherent mechanistic framework linking ubiquitination to SG homeostasis. Limitations of the study include the exclusive use of a single model system (U2OS cells), which may limit generalizability. Additionally, some observations-such as MKRN2-dependent ubiquitination within SGs and changes in DRIP accumulation under different conditions-would benefit from orthogonal validation experiments (e.g., Western blotting, immunoprecipitation, or in vitro assays) to confirm and strengthen these findings. Addressing these points would enhance the robustness and broader applicability of the conclusions.

Advance: compare the study to the closest related results in the literature or highlight results reported for the first time to your knowledge; does the study extend the knowledge in the field and in which way? Describe the nature of the advance and the resulting insights (for example: conceptual, technical, clinical, mechanistic, functional,...).

• The closest related result in literature is - Yang, Cuiwei et al. "Stress granule homeostasis is modulated by TRIM21-mediated ubiquitination of G3BP1 and autophagy-dependent elimination of stress granules." Autophagy vol. 19,7 (2023): 1934-1951. doi:10.1080/15548627.2022.2164427 - demonstrating that TRIM21, an E3 ubiquitin ligase, catalyzes K63-linked ubiquitination of G3BP1, a core SG nucleator, under oxidative stress. This ubiquitination by TRIM21 inhibits SG formation, likely by altering G3BP1's propensity for phase separation. In contrast, the MKRN2 study identifies a different E3 (MKRN2) that regulates SG dynamics under heat stress and appears to influence both assembly and disassembly. This expands the role of ubiquitin ligases in SG regulation beyond those previously studied (like TRIM21).

• Gwon and colleagues (Gwon Y, Maxwell BA, Kolaitis RM, Zhang P, Kim HJ, Taylor JP. Ubiquitination of G3BP1 mediates stress granule disassembly in a context-specific manner. Science. 2021;372(6549):eabf6548. doi:10.1126/science.abf6548) have shown that K63-linked ubiquitination of G3BP1 is required for SG disassembly after heat stress. This ubiquitinated G3BP1 recruits the segregase VCP/p97, which helps extract G3BP1 from SGs for disassembly. The MKRN2 paper builds on this by linking UBA1-dependent ubiquitination and MKRN2's activity to SG disassembly. Specifically, they show MKRN2 knockdown affects disassembly, and suggest MKRN2 helps prevent accumulation of defective ribosomal products (DRiPs) in SGs, adding a new layer to the ubiquitin-VCP model.

• Ubiquitination's impact is highly stress- and context-dependent (different chain types, ubiquitin linkages, and recruitment of E3s). The MKRN2 work conceptually strengthens this idea: by showing that MKRN2's engagement with SGs depends on active ubiquitination via UBA1, and by demonstrating functional consequences (SG dynamics + DRIP accumulation), the study highlights how cellular context (e.g., heat stress) can recruit specific ubiquitin ligases to SGs and modulate their behavior.

• There is a gap in the literature: very few (if any) studies explicitly combine the biology of DRIPs, stress granules, and E3 ligase mediated ubiquitination, especially in mammalian cells. There are relevant works about DRIP biology in stress granules, but those studies focus on chaperone-based quality control, not ubiquitin ligase-mediated ubiquitination of DRIPs. This study seems to be one of the first to make that connection in mammalian (or human-like) SG biology. A work on the plant DRIP-E3 ligase TaSAP5 (Zhang N, Yin Y, Liu X, et al. The E3 Ligase TaSAP5 Alters Drought Stress Responses by Promoting the Degradation of DRIP Proteins. Plant Physiol. 2017;175(4):1878-1892. doi:10.1104/pp.17.01319 ) shows that DRIPs can be directly ubiquitinated by E3s in other biological systems - which supports the plausibility of the MKRN2 mechanism, but it's not the same context.

• A very recent review (Yuan, Lin et al. "Stress granules: emerging players in neurodegenerative diseases." Translational neurodegeneration vol. 14,1 22. 12 May. 2025, doi:10.1186/s40035-025-00482-9) summarizes and reinforces the relationship among SGs and the pathogenesis of different neurodegenerative diseases (NDDs). By identifying MKRN2 as a new ubiquitin regulator in SGs, the current study could have relevance for neurodegeneration and proteotoxic diseases, providing a new candidate to explore in disease models.

Audience: describe the type of audience ("specialized", "broad", "basic research", "translational/clinical", etc...) that will be interested or influenced by this research; how will this research be used by others; will it be of interest beyond the specific field?

The audience for this paper is primarily specialized, including researchers in stress granule biology, ubiquitin signaling, protein quality control, ribosome biology, and cellular stress responses. The findings will also be of interest to scientists working on granulostasis, nascent protein surveillance, and proteostasis mechanisms. Beyond these specific fields, the study provides preliminary evidence linking ubiquitination to DRIP handling and SG dynamics, which may stimulate new research directions and collaborative efforts across complementary areas of cell biology and molecular biology.

• Please define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

I work in ubiquitin biology, focusing on ubiquitination signaling in physiological and disease contexts, with particular expertise in the identification of E3 ligases and their substrates across different cellular systems and in vivo models. I have less expertise in stress granule dynamics and DRiP biology, so my evaluation of those aspects is more limited and relies on interpretation of the data presented in the manuscript.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

This study identifies the E3 ubiquitin ligase Makorin 2 (MKRN2) as a novel regulator of stress granule (SG) dynamics and proteostasis. Using APEX proximity proteomics, the authors demonstrate that inhibition of the ubiquitin-activating enzyme UBA1 with TAK243 alters the SG proteome, leading to depletion of several E3 ligases, chaperones, and VCP cofactors. Detailed characterization of MKRN2 reveals that it localizes to SGs in a ubiquitination-dependent manner and is required for proper SG assembly, coalescence, and disassembly. Functionally, MKRN2 prevents the accumulation of defective ribosomal products (DRiPs) within SGs, thereby maintaining granulostasis. The study provides compelling evidence that ubiquitination, mediated specifically by MKRN2, plays a critical role in surveilling stress-damaged proteins within SGs and maintaining their dynamic liquid-like properties. Major issues: 1. Figures 1-2: Temporal dynamics of ubiquitination in SGs. The APEX proteomics was performed at a single timepoint (90 min heat stress), yet the live imaging data show that SG dynamics and TAK243 effects vary considerably over time: • The peak or SG nucleation was actually at 10-30 min (Figure 1B). • TAK243 treatment causes earlier SG nucleation (Figure 1B) but delayed disassembly (Figure 1A-B, D). A temporal proteomic analysis at multiple timepoints (e.g., 30 min, 60 min, 90 min of heat stress, and during recovery) would reveal whether MKRN2 and other ubiquitination-dependent proteins are recruited to SGs dynamically during the stress response. It would also delineate whether different E3 ligases predominate at different stages of the SG lifecycle. While such experiments may be beyond the scope of the current study, the authors should at minimum discuss this limitation and acknowledge that the single-timepoint analysis may miss dynamic changes in SG composition. *

Authors: We thank the reviewer for identifying this caveat in our methodology. We now discuss this limitation and acknowledge that the single-timepoint analysis may miss dynamic changes in SG composition.

* Figures 2D-E, 3G: MKRN2 localization mechanism requires clarification. The authors demonstrate that MKRN2 localization to SGs is dependent on active ubiquitination, as TAK243 treatment significantly reduces MKRN2 partitioning into SGs (Figure 2D-E). However, several mechanistic questions remain: • Does MKRN2 localize to SGs through binding to ubiquitinated substrates within SGs, or does MKRN2 require its own ubiquitination activity to enter SGs? • The observation that MKRN2 overexpression increases SG ubiquitin content (Figure 3G-H) could indicate either: (a) MKRN2 actively ubiquitinates substrates within SGs, or (b) MKRN2 recruitment brings along pre-ubiquitinated substrates from the cytoplasm. • Is MKRN2 localization to SGs dependent on its E3 ligase activity? A catalytically inactive mutant of MKRN2 would help distinguish whether MKRN2 must actively ubiquitinate proteins to remain in SGs or whether it binds to ubiquitinated proteins independently of its catalytic activity. The authors should clarify whether MKRN2's SG localization depends on its catalytic activity or on binding to ubiquitinated proteins, as this would fundamentally affect the interpretation of its role in SG dynamics. *

Authors: We thank the reviewer for this experimental suggestion. We will perform an analysis of the SG partitioning coefficient between WT-MKRN2 and a RING mutant of MKRN2.

* Figures 3-4: Discrepancy between assembly and disassembly phenotypes. MKRN2 knockdown produces distinct phenotypes during SG assembly versus disassembly. During assembly: smaller, more numerous SGs that fail to coalesce (Figure 3A-E), while during disassembly: delayed SG clearance (Figure 4A-D). These phenotypes may reflect different roles for MKRN2 at different stages, but the mechanism underlying this stage-specificity is unclear: • Does MKRN2 have different substrates or utilize different ubiquitin chain types during assembly versus disassembly? • The increased SG liquidity upon MKRN2 depletion (Figure 3F) seems paradoxical with delayed disassembly- typically more liquid condensates disassemble faster. The authors interpret this as decreased coalescence into "dense and mature SGs," but this requires clarification. • How does prevention of DRiP accumulation relate to the assembly defect? One would predict that DRiP accumulation would primarily affect disassembly (by reducing liquidity), yet MKRN2 depletion impacts both assembly dynamics and DRiP accumulation. The authors should discuss how MKRN2's role in preventing DRiP accumulation mechanistically connects to both the assembly and disassembly phenotypes. *

Authors: We thank the reviewer and will add to the Discussion a mention of a precedent for this precise phenotype from our previous work (Seguin et al., 2014).

* Figure 5: Incomplete characterization of MKRN2 substrates. While the authors convincingly demonstrate that MKRN2 prevents DRiP accumulation in SGs (Figure 5C-D), the direct substrates of MKRN2 remain unknown. The authors acknowledge in the limitations that "the direct MKRN2 substrates and ubiquitin-chain types (K63/K48) are currently unknown." However, several approaches could strengthen the mechanistic understanding: • Do DRiPs represent direct MKRN2 substrates? Co-immunoprecipitation of MKRN2 followed by ubiquitin-chain specific antibodies (K48 vs K63) could reveal whether MKRN2 mediates degradative (K48) or non-degradative (K63) ubiquitination. *

Authors: The DRiPs generated in the study represent truncated versions of all the proteins that were in the process of being synthesized by the cell at the moment of the stress, and therefore include both MKRN2 specific substrates and MKRN2 independent substrates. Identifying specific MKRN2 substrates, while interesting as a new research avenue, is not within the scope of the present study.

• * Given that VCP cofactors (such as UFD1L, PLAA) are depleted from SGs upon UBA1 inhibition (Figure 2C) and these cofactors recognize ubiquitinated substrates, does MKRN2 function upstream of VCP recruitment? Testing whether MKRN2 depletion affects VCP cofactor localization to SGs would clarify this pathway. * Authors: We thank the reviewer for requesting and will address it by performing MKRN2 KD, VCP immunofluorescence microscopy and perform SG partition coefficient analysis.

• * The authors note that MKRN2 knockdown produces a phenotype reminiscent of VCP inhibition-smaller, more numerous SGs with increased DRiP partitioning. This similarity suggests MKRN2 may function in the same pathway as VCP. Direct epistasis experiments would strengthen this connection. * Authors: This study is conditional results of the above study. If VCP partitioning to SGs is reduced upon MKRN2 KD, which we do not know at this point, then MKRN2/VCP double KD experiment will be performed to strengthen this connection.

* Alternative explanations for the phenotype of delayed disassembly with TAK243 or MKRN2 depletion- the authors attribute this to DRiP accumulation, but TAK243 affects global ubiquitination. Could impaired degradation of other SG proteins (not just DRiPs) contribute to delayed disassembly? Does proteasome inhibition (MG-132 treatment) phenocopy the MKRN2 depletion phenotype? This would support that MKRN2-mediated proteasomal degradation (via K48 ubiquitin chains) is key to the phenotype. *

Authors: We are happy to provide alternative explanations in the Discussion in line with Reviewer #2 suggestion. The role of the proteosome is out of the scope of our study.

• Comparison with other E3 ligases (Supplementary Figure 5): The authors show that CNOT4 and ZNF598 depletion also affect SG dynamics, though to lesser extents than MKRN2. However: • Do these E3 ligases also prevent DRiP accumulation in SGs? Testing OP-puro partitioning in CNOT4- or ZNF598-depleted cells would reveal whether DRiP clearance is a general feature of SG-localized E3 ligases or specific to MKRN2. *

• * Are there redundant or compensatory relationships between these E3 ligases? Do double knockdowns have additive effects? * Authors: Our paper presents a study of the E3 ligase MKRN2. Generalizing these observations to ZNF598, CNOT4 and perhaps an even longer list of E3s, may be an interesting question, outside the scope of our mission.

• * The authors note that MKRN2 is "the most highly SG-depleted E3 upon TAK243 treatment"-does this mean MKRN2 has the strongest dependence on active ubiquitination for its SG localization, or simply that it has the highest basal level of SG partitioning? * Authors: We thank the reviewer for this smart question. MKRN2 has the strongest dependence on active ubiquitination as we now clarify better in the Results.

*Reviewer #2 (Significance (Required)):

This is a well-executed study that identifies MKRN2 as an important regulator of stress granule dynamics and proteostasis. The combination of proximity proteomics, live imaging, and functional assays provides strong evidence for MKRN2's role in preventing DRiP accumulation and maintaining granulostasis. However, key mechanistic questions remain, particularly regarding MKRN2's direct substrates, the ubiquitin chain types it generates, and how its enzymatic activity specifically prevents DRiP accumulation while promoting both SG coalescence and disassembly. Addressing the suggested revisions, particularly those related to MKRN2's mechanism of SG localization and substrate specificity, would significantly strengthen the manuscript and provide clearer insights into how ubiquitination maintains the dynamic properties of stress granules under proteotoxic stress.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

In this paper, Amzallag et al. investigate the relationship between ubiquitination and the dynamics of stress granules (SGs). They utilize proximity ligation coupled mass spectrometry to identify SG components under conditions where the proteasome is inhibited by a small drug that targets UBiquitin-like modifier Activating enzyme 1 (UBA1), which is crucial for the initial step in the ubiquitination of misfolded proteins. Their findings reveal that the E3 ligase Makorin2 (MKRN2) is a novel component of SGs. Additionally, their data suggest that MKRN2 is necessary for processing damaged ribosome-associated proteins (DRIPs) during heat shock (HS). In the absence of MKRN2, DRIPs accumulate in SGs, which affects their dynamics. Major comments: Assess the knockdown efficiency (KD) for CNOT1, ZNF598, and MKRN2 to determine if the significant effect observed on SG dynamics upon MKRN2 depletion is due to the protein's function rather than any possible differences in KD efficiency. *

Authors: To address potential variability in knockdown efficiency, we will quantify CNOT4, ZNF598, and MKRN2 mRNA levels by RT-qPCR following siRNA knockdown.

* Since HS-induced stress granules (SGs) are influenced by the presence of TAK-243 or MKRN2 depletion, could it be that these granules become more mature and thus acquire more defective ribosomal products (DRIPs)? Do HS cells reach the same level of DRIPs, as assessed by OP-Puro staining, at a later time point? *

Authors: an interesting question. Mateju et al. carefully characterized the time course of DRiP accumulation in stress granules during heat shock, decreasing after the 90 minutes point (Appendix Figure S7; 10.15252/embj.201695957). We therefore interpret DRiP accumulation in stress granules following TAK243 treatment as a pathological state, reflecting impaired removal and degradation of DRiPs, rather than a normal, more “mature” stress granule state.

* Incorporating OP-Puro can lead to premature translation termination, potentially confounding results. Consider treating cells with a short pulse (i.e., 5 minutes) of OP-Puro just before fixation. *

Authors: Thank you for this suggestion. Treating the cell with a short pulse of OP-Puro just before fixation will lead to the labelling of a small amount of proteins, likely undetectable using conventional microscopy or Western blotting. Furthermore, it will lead to the unwanted labeling of stress responsive proteins that are translated with non canonical cap-independent mechanisms upon stress.

* Is MKRN2's dependence limited to HS-induced SGs? *

Authors: We will test sodium arsenite–induced stress and use immunofluorescence at discrete time points to assess whether the heat shock–related observations generalize to other stress types.

*

Minor comments: Abstract: Introduce UBA1. Introduction: The reference [2] should be replaced with 25719440. Results: Line 70, 'G3BP1 and 2 genes,' is somewhat misleading. Consider rephrasing into 'G3BP1 and G3BP2 genes'. Line 103: considers rephrasing 'we orthogonally validated the ubiquitin-dependent interaction' to 'we orthogonally validated the ubiquitin-dependent stress granule localization'. Line 125: '(fig.3C, EI Supplementary fig. 3)' Remove 'I'. Methods: line 260: the reference is not linked (it should be ref. [26]). Line 225: Are all the KDs being performed using the same method? Please specify. *

Authors: The text has been altered to reflect the reviewer’s suggestions.

*Fig.2C: Consider adding 'DEPLETED' on top of the scheme.

Reviewer #3 (Significance (Required)):

The study offers valuable insights into the degradative processes associated with SGs. The figures are clear, and the experimental quality is high. The authors do not overstate or overinterpret their findings, and the results effectively support their claims. However, the study lacks orthogonal methods to validate the findings and enhance the results. For instance, incorporating biochemical and reporter-based methods to measure degradation-related intermediate products (DRIPs) would be beneficial. Additionally, utilizing multiple methods to block ubiquitination, studying the dynamics of MKRN2 on SGs, and examining the consequences of excessive DRIPs on the cell fitness of SGs would further strengthen the research. *

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #3

Evidence, reproducibility and clarity

In this paper, Amzallag et al. investigate the relationship between ubiquitination and the dynamics of stress granules (SGs). They utilize proximity ligation coupled mass spectrometry to identify SG components under conditions where the proteasome is inhibited by a small drug that targets UBiquitin-like modifier Activating enzyme 1 (UBA1), which is crucial for the initial step in the ubiquitination of misfolded proteins. Their findings reveal that the E3 ligase Makorin2 (MKRN2) is a novel component of SGs. Additionally, their data suggest that MKRN2 is necessary for processing damaged ribosome-associated proteins (DRIPs) during heat shock (HS). In the absence of MKRN2, DRIPs accumulate in SGs, which affects their dynamics.

Major comments:

Assess the knockdown efficiency (KD) for CNOT1, ZNF598, and MKRN2 to determine if the significant effect observed on SG dynamics upon MKRN2 depletion is due to the protein's function rather than any possible differences in KD efficiency. Since HS-induced stress granules (SGs) are influenced by the presence of TAK-243 or MKRN2 depletion, could it be that these granules become more mature and thus acquire more defective ribosomal products (DRIPs)? Do HS cells reach the same level of DRIPs, as assessed by OP-Puro staining, at a later time point? Incorporating OP-Puro can lead to premature translation termination, potentially confounding results. Consider treating cells with a short pulse (i.e., 5 minutes) of OP-Puro just before fixation. Is MKRN2's dependence limited to HS-induced SGs?

Minor comments:

Abstract:

Introduce UBA1. Introduction:

The reference [2] should be replaced with 25719440.

Results:

Line 70, 'G3BP1 and 2 genes,' is somewhat misleading. Consider rephrasing into 'G3BP1 and G3BP2 genes'. Line 103: considers rephrasing 'we orthogonally validated the ubiquitin-dependent interaction' to 'we orthogonally validated the ubiquitin-dependent stress granule localization'. Line 125: '(fig.3C, EI Supplementary fig. 3)' Remove 'I'. Methods:

line 260: the reference is not linked (it should be ref. [26]). Line 225: Are all the KDs being performed using the same method? Please specify.

Fig.2C: Consider adding 'DEPLETED' on top of the scheme.

Significance

The study offers valuable insights into the degradative processes associated with SGs. The figures are clear, and the experimental quality is high. The authors do not overstate or overinterpret their findings, and the results effectively support their claims. However, the study lacks orthogonal methods to validate the findings and enhance the results. For instance, incorporating biochemical and reporter-based methods to measure degradation-related intermediate products (DRIPs) would be beneficial. Additionally, utilizing multiple methods to block ubiquitination, studying the dynamics of MKRN2 on SGs, and examining the consequences of excessive DRIPs on the cell fitness of SGs would further strengthen the research.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

This study identifies the E3 ubiquitin ligase Makorin 2 (MKRN2) as a novel regulator of stress granule (SG) dynamics and proteostasis. Using APEX proximity proteomics, the authors demonstrate that inhibition of the ubiquitin-activating enzyme UBA1 with TAK243 alters the SG proteome, leading to depletion of several E3 ligases, chaperones, and VCP cofactors. Detailed characterization of MKRN2 reveals that it localizes to SGs in a ubiquitination-dependent manner and is required for proper SG assembly, coalescence, and disassembly. Functionally, MKRN2 prevents the accumulation of defective ribosomal products (DRiPs) within SGs, thereby maintaining granulostasis. The study provides compelling evidence that ubiquitination, mediated specifically by MKRN2, plays a critical role in surveilling stress-damaged proteins within SGs and maintaining their dynamic liquid-like properties.

Major issues:

1. Figures 1-2: Temporal dynamics of ubiquitination in SGs. The APEX proteomics was performed at a single timepoint (90 min heat stress), yet the live imaging data show that SG dynamics and TAK243 effects vary considerably over time:
   • The peak or SG nucleation was actually at 10-30 min (Figure 1B).
   • TAK243 treatment causes earlier SG nucleation (Figure 1B) but delayed disassembly (Figure 1A-B, D). A temporal proteomic analysis at multiple timepoints (e.g., 30 min, 60 min, 90 min of heat stress, and during recovery) would reveal whether MKRN2 and other ubiquitination-dependent proteins are recruited to SGs dynamically during the stress response. It would also delineate whether different E3 ligases predominate at different stages of the SG lifecycle. While such experiments may be beyond the scope of the current study, the authors should at minimum discuss this limitation and acknowledge that the single-timepoint analysis may miss dynamic changes in SG composition.
2. Figures 2D-E, 3G: MKRN2 localization mechanism requires clarification. The authors demonstrate that MKRN2 localization to SGs is dependent on active ubiquitination, as TAK243 treatment significantly reduces MKRN2 partitioning into SGs (Figure 2D-E). However, several mechanistic questions remain:
   • Does MKRN2 localize to SGs through binding to ubiquitinated substrates within SGs, or does MKRN2 require its own ubiquitination activity to enter SGs?
   • The observation that MKRN2 overexpression increases SG ubiquitin content (Figure 3G-H) could indicate either: (a) MKRN2 actively ubiquitinates substrates within SGs, or (b) MKRN2 recruitment brings along pre-ubiquitinated substrates from the cytoplasm.
   • Is MKRN2 localization to SGs dependent on its E3 ligase activity? A catalytically inactive mutant of MKRN2 would help distinguish whether MKRN2 must actively ubiquitinate proteins to remain in SGs or whether it binds to ubiquitinated proteins independently of its catalytic activity. The authors should clarify whether MKRN2's SG localization depends on its catalytic activity or on binding to ubiquitinated proteins, as this would fundamentally affect the interpretation of its role in SG dynamics.
3. Figures 3-4: Discrepancy between assembly and disassembly phenotypes. MKRN2 knockdown produces distinct phenotypes during SG assembly versus disassembly. During assembly: smaller, more numerous SGs that fail to coalesce (Figure 3A-E), while during disassembly: delayed SG clearance (Figure 4A-D). These phenotypes may reflect different roles for MKRN2 at different stages, but the mechanism underlying this stage-specificity is unclear:
   • Does MKRN2 have different substrates or utilize different ubiquitin chain types during assembly versus disassembly?
   • The increased SG liquidity upon MKRN2 depletion (Figure 3F) seems paradoxical with delayed disassembly- typically more liquid condensates disassemble faster. The authors interpret this as decreased coalescence into "dense and mature SGs," but this requires clarification.
   • How does prevention of DRiP accumulation relate to the assembly defect? One would predict that DRiP accumulation would primarily affect disassembly (by reducing liquidity), yet MKRN2 depletion impacts both assembly dynamics and DRiP accumulation. The authors should discuss how MKRN2's role in preventing DRiP accumulation mechanistically connects to both the assembly and disassembly phenotypes.
4. Figure 5: Incomplete characterization of MKRN2 substrates. While the authors convincingly demonstrate that MKRN2 prevents DRiP accumulation in SGs (Figure 5C-D), the direct substrates of MKRN2 remain unknown. The authors acknowledge in the limitations that "the direct MKRN2 substrates and ubiquitin-chain types (K63/K48) are currently unknown." However, several approaches could strengthen the mechanistic understanding:
   • Do DRiPs represent direct MKRN2 substrates? Co-immunoprecipitation of MKRN2 followed by ubiquitin-chain specific antibodies (K48 vs K63) could reveal whether MKRN2 mediates degradative (K48) or non-degradative (K63) ubiquitination.
   • Given that VCP cofactors (such as UFD1L, PLAA) are depleted from SGs upon UBA1 inhibition (Figure 2C) and these cofactors recognize ubiquitinated substrates, does MKRN2 function upstream of VCP recruitment? Testing whether MKRN2 depletion affects VCP cofactor localization to SGs would clarify this pathway.
   • The authors note that MKRN2 knockdown produces a phenotype reminiscent of VCP inhibition-smaller, more numerous SGs with increased DRiP partitioning. This similarity suggests MKRN2 may function in the same pathway as VCP. Direct epistasis experiments would strengthen this connection.
5. Alternative explanations for the phenotype of delayed disassembly with TAK243 or MKRN2 depletion- the authors attribute this to DRiP accumulation, but TAK243 affects global ubiquitination. Could impaired degradation of other SG proteins (not just DRiPs) contribute to delayed disassembly? Does proteasome inhibition (MG-132 treatment) phenocopy the MKRN2 depletion phenotype? This would support that MKRN2-mediated proteasomal degradation (via K48 ubiquitin chains) is key to the phenotype.
6. Comparison with other E3 ligases (Supplementary Figure 5): The authors show that CNOT4 and ZNF598 depletion also affect SG dynamics, though to lesser extents than MKRN2. However:
   • Do these E3 ligases also prevent DRiP accumulation in SGs? Testing OP-puro partitioning in CNOT4- or ZNF598-depleted cells would reveal whether DRiP clearance is a general feature of SG-localized E3 ligases or specific to MKRN2.
   • Are there redundant or compensatory relationships between these E3 ligases? Do double knockdowns have additive effects?
   • The authors note that MKRN2 is "the most highly SG-depleted E3 upon TAK243 treatment"-does this mean MKRN2 has the strongest dependence on active ubiquitination for its SG localization, or simply that it has the highest basal level of SG partitioning?

Significance

This is a well-executed study that identifies MKRN2 as an important regulator of stress granule dynamics and proteostasis. The combination of proximity proteomics, live imaging, and functional assays provides strong evidence for MKRN2's role in preventing DRiP accumulation and maintaining granulostasis. However, key mechanistic questions remain, particularly regarding MKRN2's direct substrates, the ubiquitin chain types it generates, and how its enzymatic activity specifically prevents DRiP accumulation while promoting both SG coalescence and disassembly. Addressing the suggested revisions, particularly those related to MKRN2's mechanism of SG localization and substrate specificity, would significantly strengthen the manuscript and provide clearer insights into how ubiquitination maintains the dynamic properties of stress granules under proteotoxic stress.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

Summary:

In this study, the authors used proximity proteomics in U2OS cells to identify several E3 ubiquitin ligases recruited to stress granules (SGs), and they focused on MKRN2 as a novel regulator. They show that MKRN2 localization to SGs requires active ubiquitination via UBA1. Functional experiments demonstrated that MKRN2 knockdown increases the number of SG condensates, reduces their size, slightly raises SG liquidity during assembly, and slows disassembly after heat shock. Overexpression of MKRN2-GFP combined with confocal imaging revealed co-localization of MKRN2 and ubiquitin in SGs. By perturbing ubiquitination (using a UBA1 inhibitor) and inducing defective ribosomal products (DRiPs) with O-propargyl puromycin, they found that both ubiquitination inhibition and MKRN2 depletion lead to increased accumulation of DRiPs in SGs. The authors conclude that MKRN2 supports granulostasis, the maintenance of SG homeostasis , through its ubiquitin ligase activity, preventing pathological DRiP accumulation within SGs.

Major comments:

• Are the key conclusions convincing?

The key conclusions are partially convincing. The data supporting the role of ubiquitination and MKRN2 in regulating SG condensate dynamics are coherent, well controlled, and consistent with previous literature, making this part of the study solid and credible. However, the conclusions regarding the ubiquitin-dependent recruitment of MKRN2 to SGs, its relationship with UBA1 activity, the functional impact of the MKRN2 knockdown for DRiP accumulation are less thoroughly supported. These aspects would benefit from additional mechanistic evidence, validation in complementary model systems, or the use of alternative methodological approaches to strengthen the causal connections drawn by the authors. - Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? The authors should qualify some of their claims as preliminary.

1) MKRN2 recruitment to SGs (ubiquitin-dependent): The proteomics and IF data are a reasonable starting point, but they do not yet establish that MKRN2 is recruited from its physiological localization to SGs in a ubiquitin-dependent manner. To avoid overstating this point the authors should qualify the claim and/or provide additional controls: show baseline localization of endogenous MKRN2 under non-stress conditions (which is reported in literature to be nuclear and cytoplasmatic), include quantification of nuclear/cytoplasmic distribution, and demonstrate a shift into bona fide SG compartments after heat shock. Moreover, co-localization of overexpressed GFP-MKRN2 with poly-Ub (FK2) should be compared to a non-stress control and to UBA1-inhibition conditions to support claims of stress- and ubiquitination-dependent recruitment.

2) Use and interpretation of UBA1 inhibition: UBA1 inhibition effectively blocks ubiquitination globally, but it is non-selective. The manuscript should explicitly acknowledge this limitation when interpreting results from both proteomics and functional assays. Proteomics hits identified under UBA1 inhibition should be discussed as UBA1-dependent associations rather than as evidence for specific E3 ligase recruitment. The authors should consider orthogonal approaches before concluding specificity.

3) DRiP accumulation and imaging quality: The evidence presented in Figure 5 is sufficient to substantiate the claim that DRiPs accumulate in SGs upon ubiquitination inhibition or MKRN2 depletion but to show that the event of the SGs localization and their clearance from SGs during stress is promoted by MKRN3 ubiquitin ligase activity more experiments would be needed. - Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. Yes, a few targeted experiments would strengthen the conclusions without requiring the authors to open new lines of investigation.

1) Baseline localization of MKRN2: It would be important to show the baseline localization of endogenous and over-expressed MKRN2 (nuclear and cytoplasmic) under non-stress conditions and prior to ubiquitination inhibition. This would provide a reference to quantify redistribution into SGs and demonstrate recruitment in response to heat stress or ubiquitination-dependent mechanisms.

2) Specificity of MKRN2 ubiquitin ligase activity: to address the non-specific effects of UBA1 inhibition and validate that observed phenotypes depend on MKRN2's ligase activity, the authors could employ a catalytically inactive MKRN2 mutant in rescue experiments. Comparing wild-type and catalytic-dead MKRN2 in the knockdown background would clarify the causal role of MKRN2 activity in SG dynamics and DRiP clearance.

3) Ubiquitination linkage and SG marker levels: While the specific ubiquitin linkage type remains unknown, examining whether MKRN2 knockdown or overexpression affects total levels of key SG marker proteins would be informative. This could be done via Western blotting of SG markers along with ubiquitin staining, to assess whether MKRN2 influences protein stability or turnover through degradative or non-degradative ubiquitination. Such data would strengthen the mechanistic interpretation while remaining within the current study's scope. - Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments. The experiments suggested in points 1 and 3 are realistic and should not require substantial additional resources beyond those already used in the study. - Point 1 (baseline localization of MKRN2): This involves adding two control conditions (no stress and no ubiquitination inhibition) for microscopy imaging. The setup is essentially the same as in the current experiments, with time requirements mainly dependent on cell culture growth and imaging. Overall, this could be completed within a few weeks. - Point 3 (SG marker levels and ubiquitination): This entails repeating the existing experiment and adding a Western blot for SG markers and ubiquitin. The lab should already have the necessary antibodies, and the experiment could reasonably be performed within a couple of weeks. - Point 2 (catalytically inactive MKRN2 mutant and rescue experiments): This is likely more time-consuming. Designing an effective catalytic-dead mutant depends on structural knowledge of MKRN2 and may require additional validation to confirm loss of catalytic activity. If this expertise is not already present in the lab, it could significantly extend the timeline. Therefore, this experiment should be considered only if similarly recommended by other reviewers, as it represents a higher resource and time investment.

Overall, points 1 and 3 are highly feasible, while point 2 is more substantial and may require careful planning. - Are the data and the methods presented in such a way that they can be reproduced?

Yes. The methodologies used in this study to analyze SG dynamics and DRiP accumulation are well-established in the field and should be reproducible, particularly by researchers experienced in stress granule biology. Techniques such as SG assembly and disassembly assays, use of G3BP1 markers, and UBA1 inhibition are standard and clearly described. The data are generally presented in a reproducible manner; however, as noted above, some results would benefit from additional controls or complementary experiments to fully support specific conclusions. - Are the experiments adequately replicated and statistical analysis adequate?

Overall, the experiments in the manuscript appear to be adequately replicated, with most assays repeated between three and five times, as indicated in the supplementary materials. The statistical analyses used are appropriate and correctly applied to the datasets presented. However, for Figure 5 the number of experimental replicates is not reported. This should be clarified, and if the experiment was not repeated sufficiently, additional biological replicates should be performed. Given that this figure provides central evidence supporting the conclusion that DRiP accumulation depends on ubiquitination-and partly on MKRN2's ubiquitin ligase activity-adequate replication is essential.

Minor comments:

• Specific experimental issues that are easily addressable.
  • For the generation and the validation of MKRN2 knockdown in UOS2 cells data are not presented in the results or in the methods sections to demonstrate the effective knockdown of the protein of interest. This point is quite essential to demonstrate the validity of the system used
  • In the supplementary figure 2 it would be useful to mention if the Western Blot represent the input (total cell lysates) before the APEX-pulldown or if it is the APEX-pulldown loaded for WB. There is no consistence in the difference of biotynilation between different replicates shown in the 2 blots. For example in R1 and R2 G3BP1-APX TAK243 the biotynilation is one if the strongest condition while on the left blot, in the same condition comparison samples R3 and R4 are less biotinilated compared to others. It would be useful to provide an explanation for that to avoid any confusion for the readers.
  • In Figure 2D, endogenous MKRN2 localization to SGs appears reduced following UBA1 inhibition. However, it is not clear whether this reduction reflects a true relocalization or a decrease in total MKRN2 protein levels. To support the interpretation that UBA1 inhibition specifically affects MKRN2 recruitment to SGs rather than its overall expression, the authors should provide data showing total MKRN2 levels remain unchanged under UBA1 inhibition, for example via Western blot of total cell lysates.
  • DRIPs accumulation is followed during assembly but in the introduction is highlighted the fact that ubiquitination events, other reported E3 ligases and in this study data on MKRN2 showed that they play a crucial role in the disassembly of SGs which is also related with cleareance of DRIPs. Authors could add tracking DRIPs accumulation during disassembly to be added to Figure 5. I am not sure about the timeline required for this but I am just adding as optional if could be addressed easily.
  • The authors should clarify in the text why the cutoff used for the quantification in Figure 5D (PC > 3) differs from the cutoff used elsewhere in the paper (PC > 1.5). Providing a rationale for this choice will help the reader understand the methodological consistency and ensure that differences in thresholds do not confound interpretation of the results.
  • For Figure 3G, the authors use over-expressed MKRN2-GFP to assess co-localization with ubiquitin in SGs. Given that a reliable antibody for endogenous MKRN2 is available and that a validated MKRN2 knockdown line exists as an appropriate control, this experiment would gain significantly in robustness and interpretability if co-localization were demonstrated using endogenous MKRN2. In the current over-expression system, MKRN2-GFP is also present in the nucleus, whereas the endogenous protein does not appear nuclear under the conditions shown. This discrepancy raises concerns about potential over-expression artifacts or mislocalization. Demonstrating co-localization using endogenous MKRN2 would avoid confounding effects associated with over-expression. If feasible, this would be a relatively straightforward experiment to implement, as it relies on tools (antibody and knockdown line) already described in the manuscript.

• Are prior studies referenced appropriately?

  • From line 54 to line 67, the manuscript in total cites eight papers regarding the role of ubiquitination in SG disassembly. However, given the use of UBA1 inhibition in the initial MS-APEX experiment and the extensive prior literature on ubiquitination in SG assembly and disassembly under various stress conditions, the manuscript would benefit from citing additional relevant studies to provide more specifc examples. Expanding the references would provide stronger context, better connect the current findings to prior work, and emphasize the significance of the study in relation to established literature
  • At line 59, it would be helpful to note that G3BP1 is ubiquitinated by TRIM21 through a Lys63-linked ubiquitin chain. This information provides important mechanistic context, suggesting that ubiquitination of SG proteins in these pathways is likely non-degradative and related to functional regulation of SG dynamics rather than protein turnover.
  • When citing references 16 and 17, which report that the E3 ligases TRIM21 and HECT regulate SG formation, the authors should provide a plausible explanation for why these specific E3 ligases were not detected in their proteomics experiments. Differences could arise from the stress stimulus used, cell type, or experimental conditions. Similarly, since MKRN2 and other E3 ligases identified in this study have not been reported in previous works, discussing these methodological or biological differences would help prevent readers from questioning the credibility of the findings. It would also be valuable to clarify in the Conclusion that different types of stress may activate distinct ubiquitination pathways, highlighting context-dependent regulation of SG assembly and disassembly.
  • Line 59-60: when referring to the HECT family of E3 ligases involved in ubiquitination and SG disassembly, it would be more precise to report the specific E3 ligase identified in the cited studies rather than only the class of ligase. This would provide clearer mechanistic context and improve accuracy for readers.
  • The specific statement on line 182 "SG E3 ligases that depend on UBA1 activity are RBULs" should be supported by reference.
  • Are the text and figures clear and accurate?
  • In Supplementary Figure 1, DMSO is shown in green and the treatment in red, whereas in the main figures (Figure 1B and 1F) the colours in the legend are inverted. To avoid confusion, the colour coding in figure legends should be consistent across all figures throughout the manuscript.
  • At line 79, the manuscript states that "inhibition of ubiquitination delayed fluorescence recovery dynamics of G3BP1-mCherry, relative to HS-treated cells (Figure 1F, Supplementary Fig. 6A)." However, the data shown in Figure 1F appear to indicate the opposite effect: the TAK243-treated condition (green curve) shows a faster fluorescence recovery compared to the control (red curve). This discrepancy between the text and the figure should be corrected or clarified, as it may affect the interpretation of the role of ubiquitination in SG dynamics.
  • Line 86: adjust a missing bracket
  • There appears to be an error in the legend of Supplementary Figure 3: the legend states that the red condition (MKRN2) forms larger aggregates, but both the main Figure 3C of the confocal images and the text indicate that MKRN2 (red) forms smaller aggregates. Please correct the legend and any corresponding labels so they are consistent with the main figure and the text. The authors should also double-check that the figure panel order, color coding, and statistical annotations match the legend and the descriptions in the Results section to avoid reader confusion.
  • At lines 129-130, the manuscript states that "FRAP analysis demonstrated that MKRN2 KD resulted in a slight increase in SG liquidity (Fig. 3F, Supplementary Fig. 6B)." However, the data shown in Figure 3F appear to indicate the opposite trend: the MKRN2 KD condition (red curve) exhibits a faster fluorescence recovery compared to the control (green curve). This discrepancy between the text and the figure should be corrected or clarified, as it directly affects the interpretation of MKRN2's role in SG disassembly. Ensuring consistency between the written description and the plotted FRAP data is essential for accurate interpretation.
  • At lines 132-133, the manuscript states: "Then, to further test the impact of MKRN2 on SG dynamics, we overexpressed MKRN2-GFP and observed that it was recruited to SG (Fig. 3G)." This description should be corrected or clarified, as the over-expressed MKRN2-GFP also appears to localize to the nucleus.
  • At lines 134-135, the manuscript states that the FK2 antibody detects "free ubiquitin." This is incorrect. FK2 does not detect free ubiquitin; it recognizes only ubiquitin conjugates, including mono-ubiquitinated and poly-ubiquitinated proteins. The text should be corrected accordingly to avoid misinterpretation of the immunostaining data.
  • Figure 5A suffers from poor resolution, and no scale bar is provided, which limits interpretability. Additionally, the ROI selected for the green channel (DRIPs) appears to capture unspecific background staining, while the most obvious DRIP spots are localized in the nucleus. The authors should clarify this in the text, improve the image quality if possible, and ensure that the ROI accurately represents DRIP accumulation - in SGs rather than background signal.

Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

• In the first paragraph following the APEX proteomics results, the authors present validation data exclusively for MKRN2, justifying this early focus by stating that MKRN2 is the most SG-depleted E3 ligase. However, in the subsequent paragraph they introduce the RBULs and present knockdown data for MKRN2 along with two additional E3 ligases identified in the screen, before once again emphasizing that MKRN2 is the most SG-depleted ligase and therefore the main focus of the study. For clarity and logical flow, the manuscript would benefit from reordering the narrative. Specifically, the authors should first present the validation data for all three selected E3 ligases, and only then justify the decision to focus on MKRN2 for in-depth characterization. In addition to the extent of its SG depletion, the authors may also consider providing biologically relevant reasons for prioritizing MKRN2 (e.g., domain architecture, known roles in stress responses, or prior evidence of ubiquitination-related functions). Reorganizing this section would improve readability and better guide the reader through the rationale for the study's focus.
• At lines 137-138, the manuscript states: "Together these data indicate that MKRN2 regulates the assembly dynamics of SGs by promoting their coalescence during HS and can increase SG ubiquitin content." While Figure 3G shows some co-localization of MKRN2 with ubiquitin, immunofluorescence alone is insufficient to claim an increase in SG ubiquitin content. This conclusion should be supported by orthogonal experiments, such as Western blotting, in vitro ubiquitination assays, or immunoprecipitation of SG components. Including a control under no-stress conditions would also help demonstrate that ubiquitination increases specifically in response to stress. The second part of the statement should therefore be rephrased to avoid overinterpretation, for example:"...and may be associated with increased ubiquitination within SGs, as suggested by co-localization, pending further validation by complementary assays."
• At line 157, the statement: "Therefore, we conclude that MKRN2 ubiquitinates a subset of DRiPs, avoiding their accumulation inside SGs" should be rephrased as a preliminary observation. While the data support a role for MKRN2 in SG disassembly and a reduction of DRIPs, direct ubiquitination of DRIPs by MKRN2 has not been demonstrated. A more cautious phrasing would better reflect the current evidence and avoid overinterpretation.

Significance

General assessment: provide a summary of the strengths and limitations of the study. What are the strongest and most important aspects? What aspects of the study should be improved or could be developed?

• This study provides a valuable advancement in understanding the role of ubiquitination in stress granule (SG) dynamics and the clearance of SGs formed under heat stress. A major strength is the demonstration of how E3 ligases identified through proteomic screening, particularly MKRN2, influence SG assembly and disassembly in a ubiquitination- and heat stress-dependent manner. The combination of proteomics, imaging, and functional assays provides a coherent mechanistic framework linking ubiquitination to SG homeostasis. Limitations of the study include the exclusive use of a single model system (U2OS cells), which may limit generalizability. Additionally, some observations-such as MKRN2-dependent ubiquitination within SGs and changes in DRIP accumulation under different conditions-would benefit from orthogonal validation experiments (e.g., Western blotting, immunoprecipitation, or in vitro assays) to confirm and strengthen these findings. Addressing these points would enhance the robustness and broader applicability of the conclusions.

Advance: compare the study to the closest related results in the literature or highlight results reported for the first time to your knowledge; does the study extend the knowledge in the field and in which way? Describe the nature of the advance and the resulting insights (for example: conceptual, technical, clinical, mechanistic, functional,...).

• The closest related result in literature is - Yang, Cuiwei et al. "Stress granule homeostasis is modulated by TRIM21-mediated ubiquitination of G3BP1 and autophagy-dependent elimination of stress granules." Autophagy vol. 19,7 (2023): 1934-1951. doi:10.1080/15548627.2022.2164427 - demonstrating that TRIM21, an E3 ubiquitin ligase, catalyzes K63-linked ubiquitination of G3BP1, a core SG nucleator, under oxidative stress. This ubiquitination by TRIM21 inhibits SG formation, likely by altering G3BP1's propensity for phase separation. In contrast, the MKRN2 study identifies a different E3 (MKRN2) that regulates SG dynamics under heat stress and appears to influence both assembly and disassembly. This expands the role of ubiquitin ligases in SG regulation beyond those previously studied (like TRIM21).
• Gwon and colleagues (Gwon Y, Maxwell BA, Kolaitis RM, Zhang P, Kim HJ, Taylor JP. Ubiquitination of G3BP1 mediates stress granule disassembly in a context-specific manner. Science. 2021;372(6549):eabf6548. doi:10.1126/science.abf6548) have shown that K63-linked ubiquitination of G3BP1 is required for SG disassembly after heat stress. This ubiquitinated G3BP1 recruits the segregase VCP/p97, which helps extract G3BP1 from SGs for disassembly. The MKRN2 paper builds on this by linking UBA1-dependent ubiquitination and MKRN2's activity to SG disassembly. Specifically, they show MKRN2 knockdown affects disassembly, and suggest MKRN2 helps prevent accumulation of defective ribosomal products (DRiPs) in SGs, adding a new layer to the ubiquitin-VCP model.
• Ubiquitination's impact is highly stress- and context-dependent (different chain types, ubiquitin linkages, and recruitment of E3s). The MKRN2 work conceptually strengthens this idea: by showing that MKRN2's engagement with SGs depends on active ubiquitination via UBA1, and by demonstrating functional consequences (SG dynamics + DRIP accumulation), the study highlights how cellular context (e.g., heat stress) can recruit specific ubiquitin ligases to SGs and modulate their behavior.
• There is a gap in the literature: very few (if any) studies explicitly combine the biology of DRIPs, stress granules, and E3 ligase mediated ubiquitination, especially in mammalian cells. There are relevant works about DRIP biology in stress granules, but those studies focus on chaperone-based quality control, not ubiquitin ligase-mediated ubiquitination of DRIPs. This study seems to be one of the first to make that connection in mammalian (or human-like) SG biology. A work on the plant DRIP-E3 ligase TaSAP5 (Zhang N, Yin Y, Liu X, et al. The E3 Ligase TaSAP5 Alters Drought Stress Responses by Promoting the Degradation of DRIP Proteins. Plant Physiol. 2017;175(4):1878-1892. doi:10.1104/pp.17.01319 ) shows that DRIPs can be directly ubiquitinated by E3s in other biological systems - which supports the plausibility of the MKRN2 mechanism, but it's not the same context.
• A very recent review (Yuan, Lin et al. "Stress granules: emerging players in neurodegenerative diseases." Translational neurodegeneration vol. 14,1 22. 12 May. 2025, doi:10.1186/s40035-025-00482-9) summarizes and reinforces the relationship among SGs and the pathogenesis of different neurodegenerative diseases (NDDs). By identifying MKRN2 as a new ubiquitin regulator in SGs, the current study could have relevance for neurodegeneration and proteotoxic diseases, providing a new candidate to explore in disease models.

Audience: describe the type of audience ("specialized", "broad", "basic research", "translational/clinical", etc...) that will be interested or influenced by this research; how will this research be used by others; will it be of interest beyond the specific field?

The audience for this paper is primarily specialized, including researchers in stress granule biology, ubiquitin signaling, protein quality control, ribosome biology, and cellular stress responses. The findings will also be of interest to scientists working on granulostasis, nascent protein surveillance, and proteostasis mechanisms. Beyond these specific fields, the study provides preliminary evidence linking ubiquitination to DRIP handling and SG dynamics, which may stimulate new research directions and collaborative efforts across complementary areas of cell biology and molecular biology.

Please define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

I work in ubiquitin biology, focusing on ubiquitination signaling in physiological and disease contexts, with particular expertise in the identification of E3 ligases and their substrates across different cellular systems and in vivo models. I have less expertise in stress granule dynamics and DRiP biology, so my evaluation of those aspects is more limited and relies on interpretation of the data presented in the manuscript.

https://www.bl.uk/catalogues/illuminatedmanuscripts/record.asp?MSID=7029&CollID=27&NStart=8785 2025: https://searcharchives.bl.uk/catalog/032-002029680

相干

徹 penetrating, thorough 徹底 complete, thorough 廣的 far-reaching, inclusive, thorough 廣泛的 cyclopedic, far-flung, far-ranging 深入 thorough 透徹 penetrating, thorough 透辟 incisive, penetrating, thorough 詳 full, miniature, minuscule 詳盡 full, methodic, methodical 詳細 thorough 縝 comprehensive, elaborate, thorough 縝密 thorough 週密 careful, thorough 週全 comprehensive, thorough

審查

競爭的