Figure 5

Role of PHDs in HIF-1α degradation by MSA. (A) Inhibition of PHDs activity by DMOG reversed the degradation of HIF-1α by MSA in VHL active FaDu, and VHL inactive RC2 cells. Cells were treated with 10 μM MSA and 0.5 mM DMOG alone and in combination and HIF-1α was analyzed by western blot. β-actin expression was used as a loading control. (B) Gene specific silencing of PHD2 in RC2 cells by siRNA prevented the degradation of HIF-1α by MSA. PHD2 siRNA was transfected with lipofectamine 2000 for 24 h. Cells were treated with and without 10 μM MSA for 24 h and HIF-1α was detected by western blot. β-actin expression was used as a loading control. (C) Degradation of HIF-1α by MSA is VHL independent. VHL was inhibited by siRNA in FaDu cells expressing active VHL and treated with MSA to determine the HIF-1α degradation. This experiment was done under 0.5% oxygen level to stabilize HIF-1α in FaDu cells. β-actin expression was used as a loading control.