Figure 3. Fibroblasts overexpressing PPARγ show decreased mitochondrial activity, with a shift toward glycolysis. (A) EV-control and PPARγ-overexpressing fibroblasts were maintained in normoxia or under hypoxia for 24 h and analyzed by immunoblotting, with a panel of antibodies directed against OXPHOS markers. Note that PPARγ overexpression decreases the expression of the OXPHOS complex I in normoxia, and of complexes II and III under hypoxia, relative to control cells. β-actin was used as an equal loading control. (B) EV control and PPARγ-overexpressing fibroblasts were stained with MitoTracker to visualize mitochondrial activity. MitoTracker accumulates in functional mitochondria with an active membrane potential, thus correlating with mitochondrial activity. DAPI was used to visualize nuclei (blue). Fibroblasts expressing PPARγ exhibit decreased MitoTracker staining (red), relative to EV-controls, consistent with impaired mitochondrial activity. Original magnification, 60×. (C) Lactate assay. EV-control and PPARγ-overexpressing fibroblasts were maintained under hypoxia for 12 h. Then, L-lactate concentrations were measured in the cell culture media and normalized for total cell number. Note that fibroblasts overexpressing PPARγ display a 70% increase in L-lactate accumulation, relative to control EV fibroblasts. p values are as shown.