Figure 4.

BRCA2 inactivation induces endogenous oxidative stress to trigger R-loop accumulation and diminish mtDNA replication

(A and B) Bar graph showing relative ROS levels in HeLa Kyoto cells depleted of BRCA2 with (si)RNA for 72 h (left) or in BRCA2-deficient EUFA423 cells compared to EUFA423-B2 controls complemented with wild-type BRCA2 (right). Intracellular ROS was measured either using 5 μM of CM-H2DCFDA probe incubated for 15 min (A) or with 5 μM MitoSOX probe incubated for 10 min (B). The plots show the mean ± SD from three independent experiments. The two-tailed Student’s t test was performed to determine statistical significance between the two groups. ^∗p < 0.05, ∗∗p < 0.01, and ^∗∗∗p < 0.001.

(C and D) DRIP analysis of the D-loop region in EUFA423 cells compared to EUFA423-B2 controls (C) or in HeLa Kyoto treated with siBRCA2 (D). R-loop digestion by RNaseH1 serves as a control for antibody specificity. Cells were treated where indicated with 2 mM of NAC for 16 h. Error bars indicate the mean ± SD from three independent experiments. The two-way ANOVA test was performed for all pairwise comparisons to determine statistical significance. Statistically significant differences are indicated. ^∗p < 0.05.

(E) mtDNA content recovery, in the presence or absence of NAC treatment for 4 days, measured by qPCR in HeLa Kyoto cells or their CRISPR-Cas9-engineered BRCA2 nullizygous counterparts (B2-KO), after ethidium bromide (EtBr) depletion for 4 days. The graph shows the mean ± SD from three independent experiments. The two-tailed Student’s t test was performed to determine statistical significance between the two groups at the latest time point. ^∗p < 0.05.