- Mar 2021
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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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
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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
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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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
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