Fig. 3

BBIT20 inhibits the BRCA1-BARD1 interaction, in PDAC and yeast cells. (A-C) Disruption of the BRCA1-BARD1 interaction by 12 and 18 µM of BBIT20, after (A) 8 h (in PANC-1 cells) and (B) 4 h (in MIA-PaCa-2 cells), evaluated by co-immunoprecipitation. In (A, B), representative immunoblots are shown, with whole-cell lysates represented as inputs. GAPDH was used as a loading control for inputs. In (C), quantification of BARD1 protein levels co-immunoprecipitated with BRCA1, using BRCA1 from the co-immunoprecipitation as a loading control; values with DMSO were set as 1; data are mean ± SEM of four independent experiments; values significantly different from DMSO: ****p < 0.0001 (two-way ANOVA with Dunnett’s test). In (D, E), nuclear-cytoplasmic translocation of BRCA1, after 48 h of treatment with 6 µM of BBIT20, analysed by immunofluorescence. In (D), representative images (scale bar = 50 μm; 400× magnification) of BRCA1 staining (green) with nuclear counterstaining (DAPI, blue) are shown. In (E), quantification of BRCA1 nuclear and cytoplasmic foci formation, represented as mean ± SEM of four independent experiments (100 cells per sample); values significantly different from DMSO: ****p < 0.0001 (two-way ANOVA with Sidak’s test). In (F, G), yeast two-hybrid assay showing the effect of 10 and 20 µM of BBIT20 on disrupting the BRCA1-BARD1 interaction, evaluated by measuring the growth of diploid yeast strain co-expressing BRCA1 and BARD1 on SD/-Leu/-Trp/-His/-Ade/X-alpha-Gal plates. In (F), effect of 10 and 20 µM of BBIT20 on disrupting the BRCA1-BARD1 interaction, but not murine p53-SV40 large T-antigen interaction (control), in diploid yeast strains. Representative images are shown, colony growth was assessed after 2 days of incubation at 30 ºC. In (G), quantification of colony-forming units per liter (CFUs/L) of a diploid yeast strain co-expressing BRCA1 and BARD1 treated with 10 and 20 µM of BBIT20, after 2 days of incubation at 30 ºC; data are mean ± SEM of four independent experiments; values significantly different from DMSO: **p < 0.01, ****p < 0.0001 (one-way ANOVA with Dunnett’s test)