Fig 2: Differential requirement of BRCA1/BRCA2 by OvCa cells upon estrogen exposure. a-b, Quantifications for 53BP1 or γH2AX foci in A549 (a) and L02 (b) cells after estrogen treatment (50 nM) for 6 h. No elevation of DSBs was observed in these cells. c-f, Quantifications for 53BP1 foci formation in HO-8910 (a), HO-8910 PM (b), OVCAR-8TR (c) and SKOV3 (d) cells treated for estrogen for 6 h. p values: unpaired t-test. NS: not statistically significant

Fig 3: Nuclear wild type BRCA1-BARD1 is relatively stable under oxidative conditions in HCC70 breast cancer cells.(a) 8-Oxo-guanine (8-OxoG) accumulation (red) was detected in the nuclei (blue) of HCC70 cells and pre-stressed cells (HCC70stress) after treating with 1 mM H2O2. Fluorescence microscopy detected monoclonal antibodies specific for the 8-OxoG modification. Untreated cells did not show signs of 8-OxoG accumulation during a 40-minute incubation. Scale bar is 100 μm. (b) Western blot analysis indicated BRCA2, BRCA1, and BARD1 were primarily found in the nucleus. BRCA2 nuclear levels declined upon H2O2 treatment while BRCA1 and BARD1 were relatively stable. β-actin served as a loading control. (c) Co-IP experiments showed stable interactions between BRCA1 (~220 kDa) and BARD1 (~87 kDa) in the enriched nuclear material of cells experiencing oxidative stress. RNAP II phosphorylated at Pser2 repeats (~260 kDa) served as a negative control. Species-specific IgG control experiments showed low background signal. *denotes immunoprecipitated proteins (IP); unbound/depleted material (DEP); immunoblot (IB). Please see Supplementary Fig. 2 for additional western blots.

Fig 4: Establishing a breast cancer model system to measure reactive oxygen species (ROS) and wild type BRCA1-BARD1 interactions.(a) Upon treating cells with 1 mM H2O2 for 40 minutes, ROS levels (red) were detected in whole cells using fluorescent microscopy. Scale bar is 100 μm. (b) Fluorescence readings indicated less ROS accumulation in untreated than treated cells. Results show mean ± standard deviation. (c) Western blot analysis shows wild type BRCA1 (~220 kDa) eluted from Ni-NTA agarose beads in the same fraction (E2) as BARD1 (~87 kDa). K48-linked ubiquitin moieties (K48-Ubq) in the eluted material migrated at ~220 kDa. Co-IP experiments show interactions between BRCA1 and BARD1 in the eluate, and that K48-linked ubiquitin moieties associated with BRCA1. Please see Supplementary Figs 1 and 2 for additional western blots. Nuclear extract (NE); eluted material (E); input material (IN); unbound/depleted material (DEP); immunoprecipitated proteins (IP*).

Fig 5: DSB recruitment of HR proteins upon estrogen induction. a-b, IF staining (a) and quantification (b) for phosphor-BRCA1 in cryosectioned ovarian cancer at indicated stages. p values were calculated between two indicated groups by unpaired t-test. p values: unpaired t-test. c-e, Representative images and quantifications for phosphorylated RPA (pSerine 33, c), BRCA1 (pSerine 1524, d) and RAD51 (e) upon estrogen treatment. Similar to 53BP1 foci formation, levels of these protein at DSBs were elevated upon estrogen treatment