Fig. 3

ROS production by Wt and Vdac1 ^−/− MEF. a Representative electron micrographs of mitochondria of Wt and Vdac1 ^−/− MEF incubated in normoxia (Nx) or hypoxia 1 % O₂ (Hx) for 72 h. b Immunofluorescence to COX4-2 and COX7A1 in Wt and Vdac1 ^−/− MEF in Hx for 72 h. c Respiratory control of Wt and Vdac1 ^−/− MEF. Oxygen Consumption Rate (OCR) was measured in real time with a Seahorse XF bioenergetic system for Wt and Vdac1 ^−/− MEF in Nx or Hx. ΔOCR was calculated from at least four measurements before and after treatment with rotenone at 1 μM. d Quantitative measurement of ROS production was done by staining with the fluorescent probe 2', 7'-dichlorofluorescin diacetate (DCFH-DA) followed by flow cytometry. These graphs are representative of four different experiments; p < 0.02, significant difference to Wt MEF in normoxia (Nx). e Wt and Vdac1 ^−/− MEF seeded at the same density were incubated in Nx for 3 days in the presence of N-acetyl-l-cysteine (NAC, up to 1 mM). Mean ± SEM is representative of two independent experiments carried out in duplicate. f Wt and Vdac1 ^−/− MEF were incubated in Hx for 72 h in the absence (−) or presence (+) of NAC (1 mM) and cell lysates were analyzed by immunoblotting for P-ERK. ARD1 was used as a loading control. g Wt (+) and Vdac1 ^−/− (−) MEF were incubated in Nx for 24 h in the absence (−NAC) or presence (+NAC) of NAC (1 mM) and cell lysates were analyzed by immunoblotting for HIF-1α. ARD1 was used as a loading control. h Wt and Vdac1 ^−/− MEF were incubated in Nx for 3 days in the presence of H₂O₂ (up to 100 μM). Mean ± SEM is representative of two independent experiments carried out in duplicate. i Wt (+) and Vdac1 ^−/− (−) MEF were incubated in Nx or Hx for 72 h and cell lysates were analyzed by immunoblotting to GPX7. ARD1 was the loading control