Fig. 6

Attenuation of RAD51 in animal models and human patients of pulmonary arterial hypertension. a RAD51 and α-tubulin (loading control) protein amount was measured in whole lung lysates from 6-month-old BMPR2 R899X mutant mice (R899X/+) or littermate controls (+/+). Representative image of the blot and the quantitation of four individual samples are shown (n = 4). b RAD51 protein amount in pulmonary arterial smooth muscle cells isolated from control rats (+/+) or rats with heterozygous BMPR2 W508X mutation (W508X/+) were examined. Representative image of immunoblot and the quantitation of four independent experiments are shown (n = 4). c Representative images of immunohistochemistry of RAD51 and von Willebrand factor (vWF), a marker for endothelial cells, in lungs isolated from male control and SUGEN-5416/chronic hypoxia (SU/H)-treated rats (n = 3). Arrows indicate the endothelium of pulmonary arteries. Scale bars indicate 25 μm. d Representative images of hematoxylin and eosin (H&E) and immunohistochemical staining for RAD51 (200×), vWF, and α-smooth muscle actin (αSMA), a marker for smooth muscle cells, in lungs isolated from a control individual and an idiopathic pulmonary arterial hypertension patient (IPAH) (n = 4). Asterisks and arrows indicate the location of pulmonary arteries, pulmonary endothelium, and pulmonary artery smooth muscle cells, respectively. Scale bars indicate 100 μm. e Representative images of immunofluorescence staining of DNA damage marker γH2AX (red), endothelial marker, vWF (green), and DAPI in lungs from control (Con) and SU/H-treated rats (n = 3). Schematic diagram of SU/H treatment and different time points of sampling lungs are indicated. The relative amount of γH2AX signal in the endothelium was quantitated. Scale bar indicate 50 μm. Bars represent mean ± SEM from four different experiments per conditions in (a, b), and (d), and from three different experiments per conditions in (c) and (e). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 versus respective control. Unpaired two-tail t-test was used in (a–d). One-way ANOVA followed by Tukey’s multiple comparisons test was used in (e)