Fig. 3
ONC212 failed to induce mitochondrial membrane potential (ΔѰm) loss and cytochrome c release. a. Flow cytometry histogram showing the relative fluorescence intensity of mitochondrial stain TMRM in ONC212-treated (24 h, 48 h) compared to untreated HeLa and A549 cancer cells. Insignificant reduction in TMRM intensity suggests unaltered ΔѰm in cells exposed to ONC212. b. Fluorescent microscopic images of cells stained simultaneously with TMRM and Hoechst 33,342 (20X, scale bar: 50 µm). TMRM intensity was not reduced significantly upon ONC212 treatment. However, out of total condensed nuclei, very few nuclei with massive condensation showed loss of TMRM stain. c. Cytochrome c release from mitochondria was assessed by immunofluorescence and representative images are shown for control and ONC212-treated HeLa (40X, scale bar: 50 µm and A549 (60X, scale bar: 50 µm) cancer cells. Notably, cells with condensed nuclei (DAPI stain) did not reveal a diffuse (cytosolic) pattern of cyt-c but rather a predominantly granular (mitochondrial) pattern