15 Matching Annotations
  1. Apr 2021
    1. But decentralized learning goes farther than that: in a decentralized, Collaborative Learning environment, each team member participates in the learning process. They can identify their learning needs, request courses, give feedback on existing courses, and create courses themselves. We call this a bottom-up approach
      • push vs pull for learning - create an environment that enables learning to happen, and let the people doing the work surface what they need to learn, and then help facilitate and amplify that process
    1. Leaders from Accenture and DBS Bank told Harvard Business Review that encouraging employees to teach newly-acquired skills to their colleagues expanded and deepened learning for all. The training of a single employee results in learning opportunities for dozens of others. Collaborative approaches to training ripple through an organization, where ideas and methodologies cross-pollinate from one part of the business to another

      by investing in a learning organization, and learning eco-systems, we can turn learning into an active, social collaborative activity - which can benefit everyone, adn help break down silos between departments and teams.

    1. Many companies view L&D as a service provider for employees instead of a strategic partner for growth

      I've talked about this before when brain storming on how to teach companies to become teaching organizations, and partnering more closely than one-off training that is very off the shelf.

  2. Mar 2021
    1. Homology directed repair (HDR) assayEach variant was introduced into a HA-FLAG-tagged full-length PALB2 complementary DNA (cDNA) expression in the pOZC plasmid by site-directed mutagenesis using pfu turbo. Variants were verified by Sanger sequencing. Cotransfection of PALB2 expression constructs and the I-SceI expression plasmid into B400/DR-GFP reporter cells was performed at a 5:1 molar ratio using Xtremegene 9 transfection reagent (Roche). At least two independent clones containing each variant were analyzed in duplicate. PALB2 expression and transfection efficiency was verified by western blotting. Green fluorescence protein (GFP) expressing cells were quantified by fluorescence-activated cell sorting. Fold increases in GFP-positive cells, which are equivalent to HDR fold change, were normalized and rescaled relative to a 1:5 ratio derived from the p.Y551X pathogenic variant control and the wild-type PALB2 control.

      AssayGeneralClass: BAO:0003061 reporter protein

      AssayMaterialUsed: CLO:0036938 tumor-derived 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: Homology directed repair (HDR) activity fold change, measured as GFP-positive cells and normalized relative to wild type PALB2 (set to 5.0) and the p.Y551X truncating variant (set to 1.0).

      AssayRange: scaled score

      AssayNormalRange: >4.4

      AssayAbnormalRange: ≤1.7 for "deleterious" variants and ≤2.4 for "hypomorphic"variants

      AssayIndeterminateRange: >2.4-<4.4

      ValidationControlPathogenic: 7

      ValidationControlBenign: 4

      Replication: At least 2 independent clones per variant, each analyzed in duplicate

      StatisticalAnalysisDescription: Not reported

    2. 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

    3. 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

    4. ImmunofluorescenceLive cell imaging and microirradiation studies of HeLa cells transfected with peYFP-C1-PALB2 WT or variant constructs were carried out with a Leica TCS SP5 II confocal microscope. To monitor the recruitment of YFP-PALB2 to laser-induced DNA damage sites, cells were microirradiated in the nucleus for 200 ms using a 405-nm ultraviolet (UV) laser and imaged every 30 seconds for 15 minutes. Fluorescence intensity of YFP-PALB2 at DNA damage sites relative to an unirradiated nuclear area was quantified (Supplemental Materials). Cyclin A–positive HeLa cells treated with siCtrl and siRNA against PALB2 were complemented with wild-type and mutant FLAG-tagged PALB2 expression constructs, exposed to 2 Gy of γ-IR, incubated for 6 hours, and subjected to immunofluorescence for RAD51 foci. HeLa cells were fixed with 4% (w/v) paraformaldehyde for 10 minutes at room temperature, washed with tris-buffered saline (TBS), and fixed again with ice-cold methanol for 5 minutes at −20 °C. Cells were incubated for 1 hour at room temperature with the anti-RAD51 (1:7000, B-bridge International, 70-001) and anticyclin A (1:400, BD Biosciences, 611268), and incubated for 1 hour at room temperature with the Alexa Fluor 568 goat antirabbit (Invitrogen, A-11011) and Alexa Fluor 647 goat antimouse (Invitrogen, A-21235) secondary antibodies. Z-stack images were acquired on a Leica CTR 6000 microscope and the number of RAD51 foci per cyclin A–positive cells expressing the indicated YFP-PALB2 constructs was scored with Volocity software v6.0.1 (Perkin–Elmer Improvision). Results represent the mean (± SD) of three independent trials (n = 50 cells per condition). HEK293T cells transfected with PALB2 expression constructs were also subjected to immunofluorescence for PALB2 using the monoclonal anti-FLAG M2 antibody (Sigma) and the Alexa Fluor 568 goat antimouse (Life Technologies) secondary antibody.

      AssayGeneralClass: BAO:0000450 fluorescence microscopy

      AssayMaterialUsed: CLO:0003684 HeLa cell

      AssayDescription: HeLa cells were treated with PALB2 siRNA and transfected with peYFP-PALB2 expressing PALB2 variants (or empty vector), followed by exposure to 2 Gy of γ-IR. Six 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.

      AssayRange: foci/cell

      AssayNormalRange: Not reported

      AssayAbnormalRange: Not reported

      AssayIndeterminateRange: Not reported

      ValidationControlPathogenic: 0

      ValidationControlBenign: 0

      Replication: Three independent experiments with 50 cells per condition

      StatisticalAnalysisDescription: Kruskal–Wallis test with Dunn's multiple comparison post-test

    1. 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

    2. 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

    3. 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

    1. To further assess the impact of the 5 selected VUS on PALB2, we examined whether they affected the accumulation of RAD51 at IR-induced DSBs by measuring the formation RAD51 foci.

      AssayGeneralClass: BAO:0000450 fluorescence microscopy

      AssayMaterialUsed: CLO:0003684 HeLa cell

      AssayDescription: Transient expression of wild type and variant PALB2 cDNA constructs in HeLa cells following PALB2 siRNA knockdown; exposure ionizing radiation induces DNA damage; RAD51 foci formation is measured by immunofluorescence microscopy 4 h after irradiation

      AssayReadOutDescription: Number of RAD51 foci per S-phase cell (determined by cyclin A detection)

      AssayRange: foci/cell

      AssayNormalRange: RAD51 foci numbers comparable to that of cells expressing wild type PALB2; no numeric threshold given

      AssayAbnormalRange: RAD51 foci numbers comparable to that of cells expressing empty vector; no numeric threshold given

      AssayIndeterminateRange: Not reported

      ValidationControlPathogenic: 0

      ValidationControlBenign: 0

      Replication: 3 independent experiments

      StatisticalAnalysisDescription: Not reported

    2. 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

    3. 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

    4. 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

    1. 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.