Figure 4. The recruitment of Brca1/Bard1 heterodimers to PCNA replication factories is defective in HU-treated Bard1^SF/SF and Bard1^KA/KA cells.

A) Representative images of late S phase PCNA⁺ cells. Upon immunoflourescent co-staining with antibodies specific for PCNA (red) and Bard1 (green), three distinct populations of PCNA⁺ cells were identified: 1) cells displaying ≥5 PCNA foci but no Bard1 foci (PCNA ⁺Bard1^–, top); 2) cells displaying both ≥5 PCNA foci and ≥5 Bard1 foci that were spatially non-overlapping (PCNA⁺Brca1⁺ non-co-localizing, middle); and 3) cells displaying both ≥5 PCNA foci and ≥5 Bard1 foci in which more than half of the Bard1 foci co-localize with PCNA foci (PCNA⁺Brca1⁺ co-localizing, bottom). In panels B and C, the percent co-localization is the number of PCNA⁺Bard1⁺ co-localizing cells divided by the total number of PCNA⁺ late S phase cells (PCNA⁺Bard1^– + PCNA⁺Bard1⁺ co-localizing + PCNA⁺Bard1⁺ non -co-localizing).

B) The percentage of late S phase cells with co-localizing PCNA and Bard1 foci in isogenic Bard1^+/+ and Bard1^SF/SF MEFs cultured for 90 minutes in the presence or absence of 2mM hydroxyurea (HU) and/or 100 nM olaparib (PARPi). At least 200 late S phase PCNA⁺ cells were examined for each condition. The histogram presents the average of three independent experiments and the error bars represent standard error of the mean. Statistical analyses were performed using one-way ANOVA (**** p<0.0001).

C) The percentage of late S phase cells with co-localizing PCNA and Bard1 foci in Bard1^+/+ and Bard1^KA/KA MEFs cultured in the presence or absence of HU and/or PARPi.

D) The percentage of late S phase cells with co-localizing PCNA and Brca1 foci in Bard1^+/+ and Bard1^SF/SF MEFs cultured in the presence or absence of HU and/or PARPi.

E) The percentage of late S phase cells with co-localizing PCNA and Brca1 foci in Bard1^+/+ and Bard1^KA/KA MEFs cultured in the presence or absence of HU and/or PARPi.