Journal: NPJ Breast Cancer
Article Title: Functional assessment of missense variants of uncertain significance in the cancer susceptibility gene PALB2
doi: 10.1038/s41523-022-00454-6
Figure Lengend Snippet: a Schematic representation of the HR repair assay used in our study. b HR activity of the selected PALB2 VUSs. PALB2-knockdown U2OS/DR-GFP cells were co-transfected with an I-SceI expression vector and the siRNA-resistant PALB2 constructs (or an empty vector, EV). Data represent the mean percentage ( ± SEM) of GFP-positive cells relative to the WT condition from three independent experiments. Statistical significance was analyzed by one-way ANOVA and Dunnett’s multiple comparisons test. *** P < 0.001; **** P < 0.0001. c Amino acid sequence alignment of the PALB2 coiled-coil domain from various species. PALB2 VUSs that significantly impaired the HR activity were marked on the top. d The PALB2 VUSs disrupted BRCA1-PALB2 interactions. The indicated FLAG-tagged PALB2 constructs (or an empty vector, EV) were transiently transfected in 293 T cells, and PALB2 proteins were immunoprecipitated with anti-FLAG M2 beads. PALB2-binding proteins were analyzed by western blotting. Whole cell lysate (WCL) showed levels of PALB2 expression. e Quantification of western blotting band intensities ( d ) from three independent experiments. BRCA1 and RAD51 protein expressions were normalized to PALB2 protein levels and compared to the WT condition. Data presented as mean ± SEM. Statistical significance was analyzed by one-way ANOVA and Dunnett’s multiple comparisons test. * P < 0.05; **** P < 0.0001; ns, not significant.
Article Snippet: After 48 h of transfection, 500,000 cells were collected for each condition and then co-transfected with 3 μg of I-SceI expression vector and 1.5 μg of various siRNA-resistant pOZ-FH-C1-PALB2 constructs (or pOZC empty vector) using Gene Pulser Xcell (Bio-Rad, Hercules, CA, USA).
Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Construct, Sequencing, Immunoprecipitation, Binding Assay, Western Blot
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