Fig. 4

Metabolic characteristics of Wt and Vdac1 ^−/− MEF incubated in normoxia (Nx) or hypoxia 1 % O₂ (Hx). a Wt (+) and Vdac1 ^−/− (−) MEF were incubated in Nx or Hx for 72 h and cell lysates were analyzed by immunoblotting for HIF-1α and HKII. β-tubulin was used as a loading control. The extracellular acidification rate (ECAR) in b Nx or c Hx of Wt and Vdac1 ^−/− MEF was evaluated with a Seahorse XF bioenergetic system. Cells were deprived of glucose for 1 h, then glucose (Glu 10 mM) and oligomycin (Oligo 1 μM) were injected at the indicated times. d After 3 days of culture, cells were lysed in Assay Buffer with sonication. The amount of lactate was quantified in cell extracts. The mean ± SEM is representative of three independent experiments carried out in duplicate. A p < 0.001 and p < 0.005 show significant differences. e ATP production of Wt and Vdac1 ^−/− MEF in Nx or Hx for 72 h. The mean ± SEM is representative of three independent experiments carried out in duplicate. A p < 0.0005 and p < 0.00005 show significant differences. f Wt and Vdac1 ^−/− MEF were cultured for 2 days in Nx or Hx in the presence of oligomycin (Oligo, 1 μM), metformin (−Metf), and without glutamine (−Gluta), in the presence of dialyzed serum. The cell number was measured on a Beckman Coulter apparatus (squares). The percentage cell mortality was measured by trypan blue exclusion (black dots). The mean ± SEM is representative of two independent experiments carried out in duplicate. A p < 0.01, p < 0.001, and p < 0.005 show significant differences