Figure 5.

Respiratory chain function upon PDE12 overexpression. (A) Growth of cells overexpressing PDE12 on galactose and glucose media. Growth curves of parental HEK293 cells or transfectants-expressing PDE12 or the E351A mutant in galactose or glucose (inset) media and induced for the indicated time. (B) Mitochondrial membrane potential in cells overexpressing PDE12. Mitochondrial membrane potential was assessed by a quantitative FACS analysis of TMRE intensity. Twenty thousand cells stained with TMRE were analysed for each sample. The percentages of TMRE intensity were normalized to that of HEK293T cells (=100%). The mtDNA-less HEK293T cells were used as a negative control. **P < 0.01, ***P < 0.001; two-tailed Student’s t-test; n = 3, Error bars = 1 SD. (C) Steady-state level of the OXPHOS subunits in cells overexpressing PDE12. Steady-state protein level of subunits of respiratory chain complexes in uninduced control cells or cells overexpressing PDE12 or its catalytic mutant (E351A) for the indicated time were analysed by western blot. β-Actin was used as a loading control. (D) OCR in cells overexpressing PDE12. OCR measured in an extracellular flux Seahorse instrument in a quadruplicate population of control uninduced or cells overexpressing PDE12 or its catalytic mutant (E351A) for 4 days. The wells containing cells were sequentially injected with 20 mM 2-DG to inhibit glycolysis, 100 nM oligomycin to inhibit ATP synthase, 1 µM FCCP to uncouple the respiratory chain and 200 nM rotenone to inhibit complex I.** P < 0.01; two-tailed Student’s t-test; n = 3, Error bars = 1 SD.