Figure 2.

pVHL re-expression or HIF-2α knockdown increases basal OCR. (A,B) Graphs shows routine (basal) OCR (A) and maximal respiratory capacity (B) in 786O-VHL (VHL) and 786O-EV (EV) cells. OCR (pmol.s⁻¹) was corrected for cell number (per 10⁶ cells) and non-mitochondrial respiration (n = 4). (C) Graph shows respiratory flux ratios (R/E, routine control ratio; L/E, leak control ration; R-L/E, net-routine control ratio) calculated from OCR data described in (A) (n = 4). (D) Graph shows luminescence (RLU) as a measure of total cellular ATP content in 786O-VHL (VHL) and 786O-EV (EV) cells, normalized to cell number (n = 3). (E) Graph show routine (basal) OCR and maximal respiratory capacity in 786O-EV cells transiently expressing a non-silencing control siRNA (Ctrl) or HIF-2α siRNA (HIF-2α KD). Data are presented as mean ± S.D. n = 4 (*p < 0.05) and (**p < 0.01). (F) Western blots show HIF-2α, COXIV and CHCHD4 protein levels in two independent 786O cell pools (i and ii) stably transduced with increasing amounts of control shRNA and two independent HIF-2α shRNAs(1) and (2). Data are representative of three independent experiments. (G) Graphs show relative levels of CHCHD4, NDUFB10 and COX IV protein from densitometric analysis of western data from experiments described in (F). Values were normalized to the load. Data are presented as mean ± S.D. (n.s. p > 0.05, *p < 0.05, ***p < 0.001, and ****p < 0.0001).