Fig. 6
A combination of ONC212 and Navitoclax induced insignificant mitochondrial membrane potential (ΔѰm) loss and cytochrome c release in cancer cells. a. Flow cytometry histogram showing the relative fluorescence intensity of mitochondrial stain TMRM in cells treated with ONC212 (48 h), a combination of ONC212 and Navitoclax (24 h) and etoposide (positive control). b and c. Immunofluorescence images (A549: 60X and HeLa: 40X, scale bar: 50 μm) showing cytochrome c localization in control, ONC212 (48 h) and ONC212 with navitoclax treated (24 h) A549 and HeLa cancer cells. The magnified area highlights the granular pattern of cyt-c even in the A549 cell with condensed nuclei upon treatment suggesting insignificant cyt-c release from mitochondria to cytosol. d. Fluorescence images (20X, scale bar: 100 μm) of cyt-c-GFP stably expressing HeLa cells stained with mitochondrial TMRM dye after treatment. Insignificant alternation in TMRM intensity and GFP granular pattern suggest negligible ΔѰm loss and cyt-c-GFP release after treatment. e. Confocal microscopic images of HeLa cyt-c-GFP cells stained with propidium iodide (PI) after 24 h of co-treatment with ONC212 and Navitoclax. (40X, scale bar:10 μm). Two different fields are shown. As shown by arrows, even PI positive cells reflected granular pattern of cyt-c-GFP indicating insignificant release of cyt-c-GFP in cytosol