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. 2016 Aug 6;7(36):58331–58350. doi: 10.18632/oncotarget.11110

Figure 3. Death of B16F10 cells by ASK displays parameters of apoptosis.

Figure 3

A. ASK induces dissipation of Δψm. B16F10 cells were transfected with 200 nM ASO-C or ASO-1560S, or left untreated for 24h. A parallel culture was incubated with the uncoupling drug CCCP (see Materials and Methods). Cells were then harvested at 24 h post-transfection, stained with 20 nM TMRM for 15 min and analyzed by flow cytometry. ASK and CCCP induce dissipation of Δψm. B. A triplicate analysis shows that ASO-1560S induces about 55% dissipation of Δψm compared to 95% for CCCP. C. ASK induces release of cytochrome c. B16F10 cells treated as described in (A) or incubated with 5 μm STP as positive control were harvested at 24 h post-transfection. Western blot of the cytosolic fraction (see Materials and Methods) reveals, as compared to controls, a significant release of cytochrome c to the cytosol in cells transfected with ASO-1560S or incubated with STP. D. ASK induces caspase activation. B16F10 cells treated as in (A) for 24h were labeled with FITC-VAD-fmk (see Materials and Methods). Parallel cultures were incubated with 5 μM STP as positive control for apoptosis. Only cells treated with ASO-1560S or STP exhibited green fluorescence due to caspase activation. Bars = 25 μm. E. A triplicate analysis shows that ASK and STP induce over 60% of cells with activated caspases as compared to controls (**p<0.01). Error bars represent average ± s.d. F. ASK induces an increase of sub-G1 fraction. B16F10 cells were treated as in (A), but for 48 h. A parallel culture was incubated with STP as positive control. Cells were then harvested, stained with PI and analyzed by flow cytometry. Only ASO-1560S or STP elicited a significant increase in sub-G1 DNA fraction of 13% and 6%, respectively (*p<0.005). G. ASK induces annexin V-positive cells. B16F10 cells were treated for 24 h as in (A), labeled with annexin V-Alexa Fluor 488 and analyzed by flow cytometry. A triplicate analysis shows that ASO-1560S induced about 45% annexin-positive cells as compared to controls (NT and ASO-C) (*p<0.005).