Figure 4. Cooperative regulation of HSPA12B and YAP expression and nuclear localization in endothelial cells after hypoxic challenge.

HUVECs were transfected with siRNA specific for HSPA12B (siHSPA12B) or for YAP (siYAP). Scrambled siRNA served as control (siNC). In separate experiments, HUVECs were transfected with Ad-HSPA12B or Ad-GFP. Twenty-four hours after transfection, cells were subjected to hypoxia or normoxia. The levels of HSPA12B and YAP in the cytosol (A and C) and the nucleus (B and D) were examined by Western blot (n = 3). (E–H) Endothelial cells were treated with YAP inhibitor, verteporfin (1 Mm), before the cells were subjected to hypoxia. The levels of HSPA12B and YAP in the cytosol and the nucleus were examined by Western blot (n = 3–4, E and F). GAPDH was used as cytosolic loading control and Histone3 was used as nuclear loading control. The mRNA levels of HSPA12B and YAP were assessed with qRT-PCR (n = 3) (G and H). Comparisons of data between groups were made using 1-way ANOVA followed by Tukey’s procedure. *P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with indicated groups. HUVECs, human umbilical vein endothelial cells; YAP, yes-associated protein.