Fig. 5

Targeting ERRα increases mitochondrial dysfunction leading to apoptosis dependent on the presence of p53. a Live cells were stained with MitoTracker Red CMXRos, and IF analysis was conducted to detect cytochrome c (cyto c). Enlarged panels represent selected digitally enlarged portions of parent images to enhance the visibility of mitochondrial ROS (orange dots); scale bar, 50 μm. b Cells were stained with H₂DCFDA and DAPI and analyzed by flow cytometry. ROS levels were determined by the conversion of H₂DCFDA to the highly fluorescent 2′,7′-dichlorofluorescein (DCF) as described in the “Methods” section. c The cytosolic and membrane/organelle fractions were analyzed by IB with anti-AIF, anti-Smac/Diablo, anti-cyto c, anti-α-Tubulin, and anti-VDAC1. d Live cells were stained with MitoTracker Red CMXRos, and IF analysis was conducted for AIF. Enlarged panels represent selected digitally enlarged portions of parent images to enhance the visibility of mitochondrial ROS (orange dots; scale bar, 50 μm). e IB analysis was conducted with anti-ERRα, anti-p53, anti-caspase-3, anti-cleaved caspase-3, anti-cleaved PARP, and anti-GAPDH. f Apoptotic cells were detected by flow cytometry and by using the Annexin V-FITC assay kit as described in “Methods” section. All experiments were performed using HCT-116^p53+/+ and HCT-116^p53−/− cells treated for 48 h with XCT790 (15 μM) or vehicle (DMSO). Data are shown as means ± S.D. (n = 2–3). The p value was calculated using a two-tailed Student’s t test. **p ≤ 0.01