Figure 2. Stabilization of HIF-1α and HIF-2α in HCT116 cells upon Baf treatment (0.25 μM) is associated with mitochondrial uncoupling.

(A) Western blot analysis demonstrates significant elevation in HIF-1α and HIF-2α levels in HCT116 WT cells treated with Baf for 5 h at 6% O₂. This effect is abolished by inhibiting mitochondrial respiration with AA. In the presence of DMOG, the level of HIF-1α and HIF-2α stabilization is similar in all samples, including those which are treated with Baf/AA. In the presence of CMA, HIF-1α and HIF-2α levels in WT cells are also increased significantly, yet to a lesser degree than upon Baf treatment. (B) Cellular ATP levels are reduced in WT cells treated with Baf/AA for 5 h at 6% O₂, regardless the presence of DMOG. Treatment with Baf in galactose (+) medium causes dramatic decrease in the ATP level. (C) The levels of PHD1, PHD3, VHL and ATP6V0C proteins are similar in WT and SCO2^−/− cells, while the content of PHD2 in SCO2^−/− cells is significantly lower than in WT control. Asterisks indicate statistically significant difference (*P<0.01 and **P<0.001, Student's t test) between the experimental samples and mock control (DMSO). Hash in (A) shows a significant difference between the effects of Baf and CMA.