Figure 6. MDA-MB-231 cells overexpressing PPARγ display increased autophagy and decreased mitochondrial activity. (A) MDA-MB-231 breast cancer cells were transduced with a lentiviral vector encoding PPARγ or the empty vector (EV) control. After selection, cells were subjected to immunoblot analysis with specific antibodies to validate PPARγ expression. β-actin is shown as an equal loading control. (B) PPARγ-overexpressing MDA-MB-231 cells and EV controls were subjected to starvation for 2–6 h and analyzed by immunoblotting using antibodies directed against a panel of autophagy markers. Note that PPARγ induces autophagy in breast cancer cells by upregulating the expression levels of Beclin-1, BNIP3, cathepsin B, Lamp1 and LC3. β-actin is shown as an equal loading control. (C) EV control and PPARγ-overexpressing MDA-MB-231 cells were maintained under hypoxia for 12 h and analyzed by immunoblotting with a panel of antibodies directed against OXPHOS complex markers. Note that PPARγ decreases the expression of OXPHOS complexes I–V in breast cancer cells, suggesting a severe impairment in mitochondrial respiration. β-actin is shown as an equal loading control. (D) EV control and PPARγ-overexpressing MDA-MB-231 cells were maintained under hypoxia for 12 or 24 h and stained with MitoTracker (red) to visualize mitochondrial activity. Nuclei were stained with DAPI (blue). Note that cancer cells harboring PPARγ show reduced MitoTracker staining, relative to control cells, indicating decreased mitochondrial activity. Original magnification, 60×.