Fig. 5
Overexpression of HSOD1-G93A in N2A cells reproduces complex I function loss, preventable by estradiol treatment. Neither cell viability a nor ATP content b was altered as a consequence of transfection or estradiol treatment. c O2 flux measurements in intact N2A cells pretreated with E2 and transfected with pEGFP-HSOD1-G93A and pEGFP-hSOD-wt showed a reduction in the maximum oxygen capacity in G93A transfected cells which was abrogated by estradiol treatment. d O2 flux measurements in above-mentioned permeabilized cells showed a complex I impairment for cells transfected with G93A with respect to control cells which was abolished by the estradiol treatment. In the same experimental regime, western blot analysis e for mitochondrial complex protein expression (complex I and complex II) confirmed an unaltered representative peptide content across different treatment and transfects. Bars in a represent % of survival ± SEM respect to control cells for 4-6 independent experiments. Bars in b represented ATP mean values ± SEM expressed as nmol/mg protein for 4-6 independent experiments. O2 consumption in b was measured at 37 °C in culture medium containing 10 mM Hepes, pH 7.4, and corrected for instrumental background O2 flux. In d culture medium was MIR05. Values were normalized for actual protein content in the respirometer chambers, expressed as pmol O2/min · mg protein. *, P < 0.05; **P < 0.01; ***P < 0.001 between control cells and G93A cells for 4-6 independent experiments. Bars in e represent means ± SEM densitometric analysis of the corresponding complexes relativized for actual porin content in each lane for 4-6 independent experiments