Figure 6.

RBM6 promotes HR of DSBs via regulating the expression of Fe65. (A) RBM6 depletion reduces the expression of RAD51, RAD54B and XRCC2 genes. RNA was extracted from MCF10A control or RBM6-KO cells and analyzed by real-time RT-qPCR to detect the mRNA levels of the indicated genes. (B) Western blot analysis shows that RBM6 depletion reduces Fe65, Rad51 and H4K16ac levels. H3 and β-actin were used as loading controls. Band intensities of Fe65, Rad51 were normalized to the intensities of their respective β-actin bands. H4K16ac bands intensities were normalized to the intensities of their respective H3 bands. The mean normalized ratio ± SD (n = 3) is shown at the bottom of the blot. Two-tailed t-test: P-value(Fe65) = 0.0057; P-value(Rad51) = 0.0017; P-value(H4K16ac) = 0.0010. (C) Western blot shows myc-Fe65 expression in RBM6-deficient MCF10A expressing myc-Fe65. β-Actin was used as a loading control. (D) Fe65 expression rescues the deficiency in HR of DSBs seen in RBM6 depleted cells. RBM6 deficient cells were transfected with vector expressing myc- Fe65 and HR efficiency was measured as described in Figure 2C. (E) MCF7 cells expressing MR-Fe65 or MR-RBM6 show elevated HR. HR efficiency was measured as in Figure 2C. Caffeine was used as a positive control. Data presented are mean of three independent experiments ± SD. *P< 0.01, **P < 0.001, ***P < 0.0001, ^****P < 0.00001. The positions of molecular weight markers are indicated to the left of all western blots.