Fig. 3.

Maintenance of RAD51 and genome integrity is controlled by BMPR2 signaling pathway. Pulmonary microvascular endothelial cells (PMVECs) were transfected with Control (Con), BMPR2, or RAD51 siRNAs. After 24 h, PMVECs were treated with mitomycin C (MMC) for 14 h or camptothecin (CPT) for 6 h. a The amount of RAD51 protein normalized to GAPDH was measured in PMVECs transfected with Con or BMPR2 siRNAs and treated with MMC or vehicle (H₂O, Con) for 14 h. Representative image and the quantitation of three independent experiments are shown (n = 3). b Single-cell gel electrophoresis (alkaline comet assay) was performed using Con or BMPR2 siRNA-transfected PMVECs after vehicle (Con) or MMC treatment for 14 h and the fraction (%) of cells with DNA damage was quantitated by ImageJ software. Representative images of alkaline comet assay and the quantitation of 60–70 cells are shown. c Alkaline comet assay was performed using Con or BMPR2 siRNA-transfected PMVECs after vehicle (DMSO, Con) or CPT treatment for 6 h and the degree of DNA damage in cells was examined. Representative images and the quantitation of 70–100 cells are shown. d Alkaline comet assay was performed using Con or RAD51 siRNA-transfected PMVECs after vehicle (Con) or CPT treatment for 6 h and the degree of DNA damaged cells was compared (n = 100–160). e Con or RAD51 siRNA was transfected in PMVECs, followed by quantitation of BMPR2 mRNA and BMPR2 protein (n = 3). GAPDH was used for normalization. Bars represent mean ± SEM from three different experiments per conditions in (a–e). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus a respective control. One-way ANOVA followed by Tukey’s multiple comparisons test was used in (a–d). Unpaired two-tail t-test was used in (e)