Figure 4.

CSC toxicity effect on mitochondrial respiration and reactive oxygen species production in cultured iMEFs. (A) Oxygen consumption of permeabilized iMEFs in respiration buffer after culturing in gal media for 48 hours conditioned with CSC as indicated. Rotenone-sensitive oxygen consumption in the presence of complex I (cI) substrates pyruvate, glutamate, and malate plus ADP. (B) Antimycin A- plus n-propyl gallate–sensitive oxygen consumption in the presence of succinate (complex II [cII]) plus rotenone. (C) Sodium azide–sensitive oxygen consumption in the presence of ascorbate/TMPD (complex IV [cIV]). (D) Representative Western blot (n ≥ 3) probed for subunits of the complexes of the mitochondrial OXPHOS system as indicated, with AOX and α-tubulin as the loading control. For entire blots, including molecular-weight markers for cropped Western blot bands, please refer to Figure E6. (E) Mitochondrial superoxide production measured using MitoSOX Red in iMEFs grown in glucose media (10 mM) after 3 hours of CSC exposure as indicated. (F) Determination of superoxide production using the spin probe CMH (1-hydroxy3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine) and subtraction of pSOD (superoxide dismutase)-sensitive signal from total signal [CMH − (CMH + pSOD)] in iMEFs grown in glucose media (10 mM) after 3 hours of CSC exposure as indicated. All data are shown as mean ± SEM; *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001 by two-way ANOVA. ESR = electronic spin resonance.