Fig. 3.

FG4592 exerts a neuroprotective effect in a HIF-α independent manner in vitro after I/R injury. A and B Representative immunoblot and analysis of HIF-1ɑ in HT-22 cells transfected with con siRNA or Hif-1α siRNA for 48 hours and further treated with FG4592 after OGD. Data are presented as the mean ± SEM, Con siRNA vs Con siRNA+FG4592: ***p < 0.001, Con siRNA+FG4592 vs Hif-1α siRNA or Hif-1α siRNA+FG4592: ###p < 0.001 (one-way ANOVA followed by Dunnett’s post-hoc test, n = 3). C The cell viability of HT-22 cells was measured by the absorbance of formazan products following OGD/R. Results are presented as mean ± SEM from four independent experiments, Con siRNA vs Con siRNA+FG4592 or Hif-1α siRNA: ***p < 0.001, *p < 0.05. Con siRNA+FG4592 vs Hif-1α siRNA: ###p < 0.001. Hif-1α siRNA vs Hif-1α siRNA+FG4592: †††p < 0.001 (one-way ANOVA followed by Dunnett’s post-hoc test). D and E Immunoblot and analysis of HIF-1ɑ in HT-22 cells. The cells of YC-1 groups were pretreated with YC-1 before 1 hour of OGD/R. All experimental groups underwent OGD for 3 hours and were treated with vehicle or FG4592 for 6 hours after reperfusion. F and G The relative expressions of Glut1 and Epo mRNA in HT-22 cells after OGD/R. The cells of PT-2385 pretreated groups were first dealt with PT-2385 for 1 hour before OGD/R. All experimental groups underwent OGD/R for 3 hours and were treated with vehicle or FG4592 for 6 hours after OGD/R. Data are presented as the mean ± SEM and from three independent experiments. Con vs other groups: ***p < 0.001, **p < 0.01, vehicle vs other groups: ###p < 0.001, FG4592 or FG4592+PT2385 (20 μM) vs FG4592+PT2385 (40 μM) or PT2385 (40 μM): †††P < 0.001 (one-way ANOVA followed by Dunnett’s post-hoc test, n = 3). H The cell viability of HT-22 cells was measured by the absorbance of formazan products following OGD/R. The cells of YC-1 and/or PT-2385 pretreated groups were first dealt with YC-1 and/or PT-2385 for 1 hour before OGD/R. All experimental groups underwent OGD/R for 3 hours and were treated with vehicle or FG4592 for 6 hours after OGD/R. Results are presented as mean ± SEM from four independent experiments, Con vs vehicle: ***p < 0.001. vehicle vs other groups: ##p < 0.01, #p < 0.05. I and J Apoptotic cell rates of HT-22 cells after OGD/R were examined by Annexin V-FITC/PI-labeled flow cytometry. Data are expressed as mean ±SEM of four independent experiments. Con vs other groups: ***p < 0.001. vehicle vs other groups: ###p < 0.001 (one-way ANOVA followed by Dunnett’s post-hoc test)