Extended Data Fig. 8. Multiple mutations can cause resistance to DNA damaging agents in Brca-deficient cells.
(a-b) Difference in progression-free survival (PFS) of BRCA2- and BRCA1-mutated ovarian serous adenocarcinoma patients with standard platinum-based regimens. Data was obtained from the TCGA project. Patients were separated into PTIP low- or high-expression based on the 33rd percentile of PTIP expression z-scores. The difference between the PFS of PTIP-low versus PTIP-high was assessed by univariate Log-rank P value (P < 0.072 and P < 0.032 in a and b, respectively). Analysis included 38 tumors with BRCA1 mutations and 34 tumors with BRCA2 mutations out of 316 high-grade serous ovarian cancers that underwent whole exome sequencing. PFS curves for PTIP-low and PTIP-high expressing tumors were generated by the Kaplan-Meier method. All reported p values are two-sided.
(c) Western blot analysis for CHD4 and PTIP levels in PEO1 cells infected with shNSC and shCHD4. Tubulin is used as loading control. (d) Cell cycle profiles in PEO1 cells infected with shNSC and shCHD4 as measured by the incorporation of EdU (y-axis) vs DAPI (x-axis). (e) Immunostaining for MRE11 and PCNA in PEO1 cells infected with shNSC and shCHD4 upon treatment with 4 mM HU. Lower panel shows the quantification for MRE11 recruitment upon HU treatment. At least 100 cells were analyzed per condition; experiments were repeated 3 times. (f) Western blot analysis for CHD4 and MRE11 levels in PEO1 cells infected with shNSC and shCHD4. Actin is used as loading control. (g) Percentage of EdU positive cells was analyzed 20 hr after Brca2−/− B cells were treated with mirin alone, mirin+PARPi or mirin+cisplatin (ns, not significant). EdU was pulsed for 20 min prior to FACS analysis. Experiments were repeated 3 times. (h) Genomic instability measured in metaphase spreads from B cells derived from Brca2−/− mice pretreated with 25 µM mirin for 2 hr followed by overnight with 1 µM PARPi or 0.5 µM cisplatin (ns, not significant, *P ≤ 0.05, ** P ≤ 0.001, Unpaired t-test). 50 metaphases were analyzed per condition. Experiments were repeated 3 times. (i) Quantification of RAD51 foci formation in WT, Brca1−/−, Parp1−/− and Brca1−/−Parp1−/− B cells treated with 10 Gy IR and harvested 4 hr post-treatment. At least 100 cells were analyzed per condition; experiments were repeated 3 times.