Figure 6.

Inhibition and silencing of HIF-2α reduces hypoxia-mediated NFATc2 nuclear translocation. (A) Immunofluorescence staining of NFATc2 in HPFs treated with HIF-1α and HIF-2α inhibitors (KC7F2, 10 µM and TC-S 7009, 50 µM, respectively) and exposed to normoxia and hypoxia (1% O₂) for 3 days. Scale Bar: 50 µm. (B) Percentages of NFATc2 nuclear-translocated cells were determined by counting the cells with NFATc2 nuclear localization signals compared to the total number of cells. (C) Lentiviral infection efficiency. BC = Blank control, VC = Vector control. (D) Western blot showing HIF-1α and HIF-2α silencing efficiency. (E) Quantitative representation of protein expression of HIF-1α and HIF-2α protein expression with HIF silencing. (F) Immunofluorescence staining of NFATc2 in HPFs treated with shRNA lentiviral constructs (MOI 100) of HIF-1α and HIF-2α and exposed to normoxia and hypoxia (1% O₂) for 3 days. Scale Bar: 50 µm. (G) Percentages of NFATc2 nuclear-translocated cells were determined by counting the cells with NFATc2 nuclear localization signals compared to the total number of cells. (H) HEK 293Ts were co-transfected with an NFAT reporter plasmid and HIF-1α or HIF-2α expression vector for 24 hrs. The reporter activities were measured as the ratio of Firefly/Renilla luciferase activities. Data represent means ± SE. *p < 0.05, **p < 0.01, ***p < 0.001 vs. normoxia or control group. n = 3 independent experiments.