Knockdown of GPR4 reduced cAMP level, CHOP expression and apoptosis in HR-treated HUVECs.

(A) HUVECs were transfected with a control or GPR4-specific siRNA. 36 h later, cells were exposed to 4 h of hypoxia, followed by 6 h of reoxygenation. Then cells were collected and subjected to immunoblotting analyses. Representative images of 6 separate experiments were shown. (B, C) Quantitative analyses of the relative levels of cleaved caspase-3 and CHOP expression. (D) The percentage of LDH release in the supernatant. (E) The intracellular cAMP level was determined. (F) Real-time RT-PCR analysis of untransfected HUVECs, scramble siRNA-transfected cells or GPR4 siRNA transfected cells. After transfection and before HR treatment, cells were harvested and mRNA levels of GPR4 were determined. Values were normalized to β-actin. The expression level of GPR4 in untransfected HUVECs was set as 1 and siRNA treatment led to about 60% reduction in target gene expression. Data were expressed as mean ± SD from four separate experiments. *P<0.05 vs. the control siRNA-transfected cells. **P<0.05 vs. the cells subjected to HR, but without NaOH or siRNA treatment.