- Mar 2021
-
www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
-
Viability assayPALB2 variants were introduced into B400 cells using mCherry-pOZC expression vector and flow cytometry for Cherry-red was performed to select for cells expressing PALB2. Sorted cells were plated in 96-well plates and exposed to increasing amounts of Olaparib or cisplatin and incubated for a period of 5 days. Presto Blue (Invitrogen) was added and incubated for 1–2 hours before measuring fluorescence intensity on a Cytation 3 microplate reader (BioTek).
AssayGeneralClass: BAO:0003009 cell viability assay
AssayMaterialUsed: CLO:0036938 tumor-derived cell line
AssayDescription: Transient expression of wild type and variant mCherry-tagged PALB2 cDNA constructs in Trp53 and Palb2-null mouse cell line; exposure to increasing concentrations of cisplatin for 5 days induces interstrand-crosslink DNA damage; cell survival is determined by measuring fluorescence intensity after staining with a cell viability reagent.
AssayReadOutDescription: Percent cell survival after treatment with cisplatin
AssayRange: %
AssayNormalRange: Cisplatin resistance levels comparable to that of cells expressing wild type PALB2; no numeric threshold given
AssayAbnormalRange: Not reported
AssayIndeterminateRange: Not reported
ValidationControlPathogenic: 0
ValidationControlBenign: 0
Replication: Not reported
StatisticalAnalysisDescription: Not reported
-
Viability assayPALB2 variants were introduced into B400 cells using mCherry-pOZC expression vector and flow cytometry for Cherry-red was performed to select for cells expressing PALB2. Sorted cells were plated in 96-well plates and exposed to increasing amounts of Olaparib or cisplatin and incubated for a period of 5 days. Presto Blue (Invitrogen) was added and incubated for 1–2 hours before measuring fluorescence intensity on a Cytation 3 microplate reader (BioTek).
AssayGeneralClass: BAO:0003009 cell viability assay
AssayMaterialUsed: CLO:0036938 tumor-derived cell line
AssayDescription: Transient expression of wild type and variant mCherry-tagged PALB2 cDNA constructs in Trp53 and Palb2-null mouse cell line; exposure to increasing concentrations of PARP inhibitor Olaparib for 5 days inhibits end-joining mediated by PARP and sensitizes cells to DNA damage; cell survival is determined by measuring fluorescence intensity after staining with a cell viability reagent.
AssayReadOutDescription: Percent cell survival after treatment with Olaparib
AssayRange: %
AssayNormalRange: Olaparib resistance levels comparable to that of cells expressing wild type PALB2; no numeric threshold given
AssayAbnormalRange: Not reported
AssayIndeterminateRange: Not reported
ValidationControlPathogenic: 0
ValidationControlBenign: 0
Replication: Not reported
StatisticalAnalysisDescription: Not reported
-
-
www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
-
CRISPR-LMNA HDR assayU2OS were seeded in 6-well plates at 200 000 cells per well. Knockdown of PALB2 was performed 6–8 h later with 50 nM siRNA using Lipofectamine RNAiMAX (Invitrogen). Twenty-four hours post-transfection, 1.5 × 106 cells were pelleted for each condition and resuspended in 100 μL complete nucleofector solution (SE Cell Line 4D-Nucleofector™ X Kit, Lonza) to which 1μg of pCR2.1-mRuby2LMNAdonor, 1 μg of pX330-LMNAgRNA2, 1 μg of the peYFP-C1 empty vector or the indicated siRNA-resistant YFP-PALB2 construct, and 150 ρmol siRNA was added. Once transferred to a 100 ul Lonza certified cuvette, cells were transfected using the 4D-Nucleofector X-unit, program CM-104, resuspended in culture media and split into 2 60-mm dishes. One dish was harvested 24 h later for protein expression analysis as described above while cells from the other were trypsinised after 48 h for plating onto glass coverslips. Coverslips were fixed with 4% paraformaldehyde and cells analyzed for expression of mRuby2-LMNA (indicative of successful HR) by fluorescence microscopy (63×) a total of 72 h post-nucleofection. Data are represented as mean relative percentages ± SD of mRuby2-positive cells over the YFP-positive population from 3 independent experiments (total n >300 YFP-positive cells per condition).
AssayGeneralClass: BAO:0003061 reporter protein
AssayMaterialUsed: CLO:0009454 U-2 OS cell
AssayDescription: U2OS cells were treated with PALB2 siRNA and synchronized to G1/S phase by double thymidine block. Cells were then co-transfected with peYFP-PALB2 expressing PALB2 variants (or empty vector), pCR2.1-mRuby2LMNAdonor, and pX330-LMNAgRNA, which generates mRuby2-Lamin A/C fusion if HDR is successful.
AssayReadOutDescription: Mean relative percentages of mRuby2-positive cells over the YFP-positive population relative to the wild type condition.
AssayRange: %
AssayNormalRange: Not reported
AssayAbnormalRange: <40%
AssayIndeterminateRange: 41%-77%
ValidationControlPathogenic: 1
ValidationControlBenign: 3
Replication: Three independent experiments, each with n > 300 YFP-positive cells per condition
StatisticalAnalysisDescription: One-way ANOVA followed by Dunnett's post hoc analysis
-
RAD51 foci assayHeLa cells were seeded on glass coverslips in 6-well plates at 225 000 cells per well. Knockdown of PALB2 was performed 18 h later with 50 nM PALB2 siRNA using Lipofectamine RNAiMAX (Invitrogen). After 5 h, cells were subjected to double thymidine block. Briefly, cells were treated with 2 mM thymidine for 18 h and release into fresh media for 9 h. Complementation using 800 ng of the peYFP-C1 empty vector or the indicated siRNA-resistant YFP-PALB2 construct was carried out with Lipofectamine 2000 during that release time. Then, cells were treated with 2 mM thymidine for 17 h and protected from light from this point on. After 2 h of release from the second block, cells were irradiated with 2 Gy and processed for immunofluorescence 4 h post-irradiation. Unless otherwise stated, all immunofluorescence dilutions were prepared in PBS and incubations performed at room temperature with intervening washes in PBS. Cell fixation was carried out by incubation with 4% paraformaldehyde for 10 min followed by 100% ice-cold methanol for 5 min at −20°C. This was succeeded by permeabilization in 0.2% Triton X-100 for 5 min and a quenching step using 0.1% sodium borohydride for 5 min. After blocking for 1 h in a solution containing 10% goat serum and 1% BSA, cells were incubated for 1 h with primary antibodies anti-RAD51 (1 :7000, B-bridge International, #70–001) and anti-cyclin A (1:400, BD Biosciences, #611268) diluted in 1% BSA. Secondary antibodies Alexa Fluor 568 goat anti-rabbit (Invitrogen, #A-11011) and Alexa Fluor 647 goat anti-mouse (Invitrogen, #A-21235) were diluted 1:1000 in 1% BSA and applied for 1 h. Nuclei were stained for 10 min with 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI) prior to mounting onto slides with 90% glycerol containing 1 mg/ml paraphenylenediamine anti-fade reagent. Z-stack images were acquired on a Leica CTR 6000 microscope using a 63× oil immersion objective, then deconvolved and analyzed for RAD51 foci formation with Volocity software v6.0.1 (Perkin-Elmer Improvision). The number of RAD51 foci per cyclin A-positive cells expressing the indicated YFP-PALB2 constructs was scored using automatic spot counting by Volocity software and validated manually. Data from three independent trials (total n = 225 cells per condition) were analyzed for outliers using the ROUT method (Q = 1.0%) in GraphPad Prism v6.0 and the remaining were reported in a scatter dot plot. Intensity values, also provided by Volocity, of 500 RAD51 foci from a representative trial were normalized to the WT mean and reported in a scatter dot plot. Horizontal lines on the plots designate the mean values.
AssayGeneralClass: BAO:0000450 fluorescence microscopy
AssayMaterialUsed: CLO:0003684 HeLa cell
AssayDescription: HeLa cells were treated with PALB2 siRNA and synchronized to G1/S phase by double thymidine block. Cells were then transfected with peYFP-PALB2 expressing PALB2 variants (or empty vector) and irradiated with 2 Gy. Four hours after irradiation, cells were subjected to immunofluorescence for RAD51 foci (where foci formation serves as marker of normal DNA damage repair function).
AssayReadOutDescription: The number of RAD51 foci per cyclin A-positive cells expressing the indicated YFP-PALB2 constructs was scored and presented as percentage change relative to the wild type mean RAD51 foci number per cell.
AssayRange: %
AssayNormalRange: Not reported
AssayAbnormalRange: Not reported
AssayIndeterminateRange: Not reported
ValidationControlPathogenic: 1
ValidationControlBenign: 3
Replication: Three independent experiments, each with 225 cells per condition
StatisticalAnalysisDescription: Kruskal–Wallis test with Dunn's multiple comparison post-test
-
Olaparib sensitivity assayFor the sensitivity assay in HeLa, 240 000 cells were seeded into one well of a six-well plate before being transfected 6–8 h later with 50 nM control or PALB2 siRNA using Lipofectamine RNAiMAX (Invitrogen). The next morning, cells were complemented with 800 ng of the peYFP-C1 empty vector or the indicated siRNA-resistant YFP-tagged PALB2 construct using Lipofectamine 2000 (Invitrogen) for 24 h and then seeded in triplicates into a Corning 3603 black-sided clear bottom 96-well microplate at a density of 3000 cells per well. The remaining cells were kept and stored at −80°C until processed for protein extraction and immunoblotting as described above. Once attached to the plate, cells were exposed to different concentrations of olaparib (Selleckchem, #S1060) ranging from 0 (DMSO) to 2.5 μM. After 3 days of treatment, nuclei were stained with Hoechst 33342 (Invitrogen) at 10 μg/ml in media for 45 min at 37°C. Images of entire wells were acquired at 4x with a Cytation 5 Cell Imaging Multi-Mode Reader followed by quantification of Hoechst-stained nuclei with the Gen5 Data Analysis Software v3.03 (BioTek Instruments). Cell viability was expressed as percentage of survival in olaparib-treated cells relative to vehicle (DMSO)-treated cells. Results represent the mean ± SD of at least 3 independent experiments, each performed in triplicate.
AssayGeneralClass: BAO:0003009 cell viability assay
AssayMaterialUsed: CLO:0003684 HeLa cell
AssayDescription: HeLa cells were treated with PALB2 siRNA followed by transfection peYFP-PALB2 expressing PALB2 variants (or empty vector) and exposed to olaparib (2.5 µM) for 3 days. Nuclei were stained with Hoechst 33342 and measured as an indicator of cell viability.
AssayReadOutDescription: Cell viability expressed as percentage of survival in olaparib-treated cells relative to vehicle (DMSO)-treated cells
AssayRange: %
AssayNormalRange: Not reported
AssayAbnormalRange: Not reported
AssayIndeterminateRange: Not reported
ValidationControlPathogenic: 1
ValidationControlBenign: 3
Replication: At least 3 independent experiments, each performed in triplicate
StatisticalAnalysisDescription: Kruskal–Wallis test with Dunn's multiple comparison post-test
-
-
www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
-
analyzed several PALB2 variants in their response to the ICL-inducing agent cisplatin
AssayGeneralClass: BAO:0002805 cell proliferation assay
AssayMaterialUsed: CLO:0037317 mouse embryonic stem cell line
AssayDescription: Stable expression of wild type and variant PALB2 cDNA constructs in Trp53 and Palb2-null mouse cell line containing DR-GFP reporter; exposure to cisplatin for 48 h induces interstrand-crosslink DNA damage; cell survival is measured by FACS 24 h after cisplatin washout
AssayReadOutDescription: Relative resistance to cisplatin represented as cell survival relative to wild type, which was set to 100%
AssayRange: %
AssayNormalRange: Cisplatin resistance levels comparable to that of cells expressing wild type PALB2; no numeric threshold given
AssayAbnormalRange: Cisplatin resistance levels comparable to that of cells expressing empty vector; no numeric threshold given
AssayIndeterminateRange: Not reported
ValidationControlPathogenic: 2
ValidationControlBenign: 2
Replication: 2 independent experiments
StatisticalAnalysisDescription: Not reported
-
sensitivity to PARPi treatment using a cellular proliferation assay
AssayGeneralClass: BAO:0002805 cell proliferation assay
AssayMaterialUsed: CLO:0037317 mouse embryonic stem cell line
AssayDescription: Stable expression of wild type and variant PALB2 cDNA constructs in Trp53 and Palb2-null mouse cell line containing DR-GFP reporter; exposure to PARP inhibitor Olaparib for 48 h inhibits end-joining mediated by PARP and sensitizes cells to DNA damage; cell survival is measured by FACS 24 h after Olaparib washout
AssayReadOutDescription: Relative resistance to PARPi represented as cell survival relative to wild type, which was set to 100%
AssayRange: %
AssayNormalRange: PARPi resistance levels comparable to that of cells expressing wild type PALB2; no numeric threshold given
AssayAbnormalRange: PARPi resistance levels ≤30% of wild type
AssayIndeterminateRange: Not reported
ValidationControlPathogenic: 12
ValidationControlBenign: 9
Replication: 2 independent experiments
StatisticalAnalysisDescription: Not reported
-
A cell-based functional assay for PALB2 variants
AssayGeneralClass: BAO:0003061 reporter protein
AssayMaterialUsed: CLO:0037317 mouse embryonic stem cell line
AssayDescription: Stable expression of wild type and variant PALB2 cDNA constructs in Trp53 and Palb2-null mouse cell line containing DR-GFP reporter; I-SceI endonuclease introduces a double-stranded break in the reporter construct and efficient repair results in GFP expression, which is detected by flow cytometry
AssayReadOutDescription: Relative homologous recombination (HR) efficiency represented as mean percentages of GFP-positive cells among the mCherry-positive cells relative to wild type, which was set to 100%
AssayRange: %
AssayNormalRange: HR levels comparable to that of cells expressing wild type PALB2; no numeric threshold given
AssayAbnormalRange: HR levels ≤40% of wild type
AssayIndeterminateRange: Not reported
ValidationControlPathogenic: 12
ValidationControlBenign: 9
Replication: 2 independent experiments
StatisticalAnalysisDescription: Not reported
-
-
www.cell.com www.cell.com
-
Automated Patch ClampingCells were patch clamped with the SyncroPatch 384PE automated patch clamping device (Nanion). To prepare cells for patch clamping, cells were washed in PBS, treated with Accutase (Millipore-Sigma) for 3 min at 37°C, then recovered in CHO-S-serum free media (GIBCO). Cells were pelleted and resuspended in divalent-free reference solution (DVF) at ∼200,000–400,000 cells/mL. DVF contained (mM) NaCl 140, KCl 4, alpha-D(+)-glucose 5, HEPES 10 (pH 7.4) adjusted with NaOH. Cells were then added to a medium resistance (4–6 MΩ) 384-well recording chamber with 1 patch aperture per well (NPC-384, Nanion), which contained DVF and internal solution: CsCl 10, NaCl 10, CsF 110, EGTA 10, HEPES 10 (pH 7.2) adjusted with CsOH. Next, to enhance seal resistance, 50% of the DVF was exchanged with a calcium-containing seal enhancing solution: NaCl 80, NMDG 60, KCl 4, MgCl2 1, CaCl2 10, alpha-D(+)-glucose 5, HEPES 10 (pH 7.4) adjusted with HCl. The cells were washed three times in external recording solution: NaCl 80, NMDG 60, KCl 4, MgCl2 1, CaCl2 2, alpha-D(+)-glucose 5, HEPES 10 (pH 7.4) adjusted with HCl. Currents elicited in response to activation, inactivation, and recovery from inactivation protocols were then recorded (Figure S2). A late current measurement was captured every 5 s. After 1 min, 50% of the external solution was exchanged with external solution containing 200 μM tetracaine hydrochloride (Sigma; effective concentration 100 μM tetracaine). After tetracaine addition, late current measurements were obtained every 5 s for 1 additional minute. At least 10 cells expressing wild-type SCN5A were included for comparison in each SyncroPatch experiment (Figure 1), and at least 2 independent transfections and at least 10 replicate cells were studied per mutant. Recordings were performed at room temperature.We also conducted experiments to assess the effects of incubation at low temperature or mexiletine (a sodium channel blocker), interventions reported to increase cell surface expression of mistrafficked channels.27Clatot J. Ziyadeh-Isleem A. Maugenre S. Denjoy I. Liu H. Dilanian G. Hatem S.N. Deschênes I. Coulombe A. Guicheney P. Neyroud N. Dominant-negative effect of SCN5A N-terminal mutations through the interaction of Na(v)1.5 α-subunits.Cardiovasc. Res. 2012; 96: 53-63Crossref PubMed Scopus (62) Google Scholar, 28Makiyama T. Akao M. Tsuji K. Doi T. Ohno S. Takenaka K. Kobori A. Ninomiya T. Yoshida H. Takano M. et al.High risk for bradyarrhythmic complications in patients with Brugada syndrome caused by SCN5A gene mutations.J. Am. Coll. Cardiol. 2005; 46: 2100-2106Crossref PubMed Scopus (99) Google Scholar, 29Pfahnl A.E. Viswanathan P.C. Weiss R. Shang L.L. Sanyal S. Shusterman V. Kornblit C. London B. Dudley Jr., S.C. A sodium channel pore mutation causing Brugada syndrome.Heart Rhythm. 2007; 4: 46-53Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 30Valdivia C.R. Ackerman M.J. Tester D.J. Wada T. McCormack J. Ye B. Makielski J.C. A novel SCN5A arrhythmia mutation, M1766L, with expression defect rescued by mexiletine.Cardiovasc. Res. 2002; 55: 279-289Crossref PubMed Scopus (77) Google Scholar, 31Valdivia C.R. Tester D.J. Rok B.A. Porter C.B. Munger T.M. Jahangir A. Makielski J.C. Ackerman M.J. A trafficking defective, Brugada syndrome-causing SCN5A mutation rescued by drugs.Cardiovasc. Res. 2004; 62: 53-62Crossref PubMed Scopus (106) Google Scholar For these experiments, cells stably expressing loss-of-function variants were generated as described above. The cells were incubated for 24 h at 30°C, or at 37°C with or without 500 μM mexiletine hydrochloride (Sigma), washed with HEK media, and were patch clamped as described above.
AssayGeneralClass: BAO:0000062 patch clamp
AssayMaterialUsed: CLO:0037237 HEK293-derived cell
AssayDescription: HEK293T-derived cells stably expressing wild type or variant SCN5A were patch clamped and currents elicited in response to activation, inactivation, and recovery from inactivation were recorded, as well as late current measurements.
AssayReadOutDescription: Peak current density relative to wild type, which was set to 100%
AssayRange: %
AssayNormalRange: Peak current density 75-125% of wild type
AssayAbnormalRange: Peak current density 10-50% of wildtype
AssayIndeterminateRange: Peak current density 50-75% of wildtype
ValidationControlPathogenic: 0
ValidationControlBenign: 10
Replication: At least 2 independent transfections and at least 10 replicate cells per variant (see ReplicateCount in FunctionalAssayResult annotations for each variant).
StatisticalAnalysisDescription: Two-tailed t tests or two-tailed Mann-Whitney U tests were used to compare variant parameters between groups of variants, while differences in dispersion between groups were tested with Levene’s test.
-
-
jmg.bmj.com jmg.bmj.com
-
We then applied the p53 functional assay on blood samples sent to our laboratory for TP53 molecular analysis (NGS screening of the 11 exons complemented by QMPSF). Molecular and functional analyses were performed in parallel, in double blind conditions.
AssayGeneralClass: BAOCL:20:0010044 targeted transcriptional assay
AssayMaterialUsed: CL:2000001 peripheral blood mononuclear cell from patients
AssayDescription: Comparative transcriptomic analysis using reverse transcription to compare peripheral blood mononuclear cells of patients with wild type or pathogenic TP53 variants in the context of genotoxic stress induced by doxorubicin treatment. p53 RNA levels were evaluated and expressed as a percentage of the mean levels obtained for the three wild-type TP53 individuals.
AdditionalDocument: PMID: 23172776
AssayReadOutDescription: The p53 mRNA levels were expressed as a ratio of the normal values obtained for 3 TP53 wild-type control individuals.
AssayRange: UO:0000187 the p53 RNA levels were evaluated and expressed as a percentage of the mean levels obtained for three wild-type TP53 individuals.
AssayNormalRange: >65%
AssayAbnormalRange: <65%
AssayIndeterminateRange: N/A
AssayNormalControl: wild type TP53
AssayAbnormalControl: LFS patient cells
ValidationControlPathogenic: 8 individuals had seven distinct TP53 variants which could be considered as likely pathogenic or pathogenic based on their ClinVar classification or their truncating nature.
ValidationControlBenign: 51 individuals had no detectable germline TP53 variant
Replication: at least two wells were seeded per patient (treated and untreated) and duplicates or triplicates were performed whenever possible.
StatisticalAnalysisDescription: Differentially expressed genes between doxorubicin-treated and untreated cells were arbitrarily defined using, as filters, a P<0.01 and fold-change cutoffs >2 or <2, for up and down regulation, respectively. The resultant signal information was analyzed using one-way analysis of variance (ANOVA, P= 0.001), assuming normality but not equal variances with a Benjamani–Hochberg correction for multiple comparisons using three groups: controls, null, and missense mutations.
SignificanceThreshold: P=0.001
Comment: statistical analysis and P value from previous publication.
-
This new quantitative assay, based on both RT-QMPSF and RT-MLPA, was first validated on 31 lymphoblastoid cell lines derived from patients with LFS harbouring different germline heterozygous TP53 variants
AssayGeneralClass: BAO:0010044 targeted transcriptional assay
AssayMaterialUsed: BTO:0000773 lymphoblastoid cell line derived from control individuals or individuals with germline TP53 variants
AssayDescription: Comparative transcriptomic analysis using RNA-Seq to compare EBV cell lines of wild type and pathogenic TP53 in the context of genotoxic stress induced by doxorubicin treatment. p53 RNA levels were evaluated and expressed as a percentage of the mean levels obtained for the three wild-type TP53 individuals.
AdditionalDocument: PMID: 23172776
AssayReadOutDescription: The p53 mRNA levels were expressed as a ratio of the normal values obtained for 3 TP53 wild-type control individuals.
AssayRange: UO:0000187 the p53 RNA levels were evaluated and expressed as a percentage of the mean levels obtained for three wild-type TP53 individuals.
AssayNormalRange: N/A
AssayAbnormalRange: N/A
AssayIndeterminateRange: N/A
AssayNormalControl: wild type TP53
AssayAbnormalControl: LFS patient cells
ValidationControlPathogenic: 8 Individuals with dominant-negative TP53 missense variants, 10 Individuals with null TP53 variants, and 13 Individuals with other TP53 missense variants
ValidationControlBenign: 3 patients with wild type TP53
Replication: experiments were performed in triplicates.
StatisticalAnalysisDescription: Differentially expressed genes between doxorubicin-treated and untreated cells were arbitrarily defined using, as filters, a P<0.01 and fold-change cutoffs >2 or <2, for up and down regulation, respectively. The resultant signal information was analyzed using one-way analysis of variance (ANOVA, P= 0.001), assuming normality but not equal variances with a Benjamani–Hochberg correction for multiple comparisons using three groups: controls, null, and missense mutations.
SignificanceThreshold: P=0.001
Comment: statistical analysis and P value from previous publication.
-