Fig. 2: Effects of PALB2 VUSs on HR activity and BRCA1 interaction.

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.