Knockdown of HIF-1α or BNIP3 did not influence Celastrol-induced cell apoptosis under hypoxia.

<p>5a. Celastrol enhanced the HIF-1α and BNIP3 expression that was accompanied by increased PARP cleavage under hypoxia. HepG2 cells were cultured under normoxia or hypoxia with or without 4 µM Celastrol for 24 h. Western blotting was used to detect protein expression. 5b. The cytotoxicity of Celastrol was enhanced under hypoxia. HepG2 cells were cultured under normoxia or hypoxia with or without 2–4 µM Celastrol for 24 h. The cells were then stained with Annexin-V/PI and analyzed by flow cytometry. The data are presented as the mean values obtained from three independent experiments. 5c. Z-VAD blocked Celastrol-induced PARP cleavage but did not affect HIF-1α accumulation. HepG2 cells were pre-treated with 10 µM zVAD for 1 h then exposed to 4 µM Celastrol for another 6 h. The proteins were detected by western blot analysis. 5d. Knockdown of HIF-1α did not affect Celastrol-induced PARP cleavage. HepG2 cells were transfected with a non-silencing siRNA or HIF-1α siRNA for 24 h, and the cells were then treated with 4 µM Celastrol for 24 h. The proteins were detected by western blot analysis. 5e, 5f. Knockdown of BNIP3 did not affect Celastrol-induced cell death. HepG2 cells were transfected with non-silencing siRNA or BNIP3 siRNA for 24 h, and the cells were then treated with 4 µM Celastrol for 24 h. Cell death was measured by Annexin-V/PI staining and flow cytometry, and the proteins were detected by western blotting.