Figure 5 | Cell Death & Disease

Figure 5

From: Essential requirement of cytochrome c release for caspase activation by procaspase-activating compound defined by cellular models

Figure 5Figure 5

Induction of mitochondrial cyt. c release, caspase activation and cell death in Bcl-2 and Bcl-xL-overexpressing cells. (a) MCFC3 vector control and Bcl-2 GFP, Bcl-xL GFP-overexpressing cells were treated with PAC-1 (75 μM) at indicated time points and stained with Hoechst to analyze nuclear condensation. Representative images are shown. The percentage of condensed nuclei relative with untreated cells are represented in graph (n=3, mean ±S.D.). MCFC3 versus MCFC3Bcl-2-GFP cells, and MCFC3 versus MCFC3Bcl-xL-GFP cells (P<0.001) for both 24 h and 36 h treatment. (b) Clonogenic survival was assessed in these cells after PAC-1 treatment. The representative images are shown. The quantitative data of clonogenic efficiency determined as described are shown. MCFC3 versus MCFC3Bcl-2-GFP cells, and MCFC3 versus MCFC3Bcl-xL-GFP cells (P<0.001). (c) Flow cytometry data showing Δψm loss in above panel of cell lines after PAC-1 treatment with indicated time points. The TMRM intensity shifting towards lefts side indicates (Δψm) loss. (d) Caspase activation was detected by western blot in these cells upon PAC-1 treatment with different time points. (e) MCFC3 vector cells were treated with PAC-1 (75 μM) for 12 h and 24 h, and immunofluorescence was done for checking cyt. c release using fluorescent microscopy. Representative cyt. c channel (red), nucleus stained with Hoechst (blue) and merged images are shown (scale bar: 10 μm). The cyt. c immunofluorescent images of MCFC3 Bcl2-GFP-overexpressing cells, and MCFC3 Bcl-xL GFP-overexpressing cells after treatment with the same drug are also shown. The percentage of cyt. c releasing cells observed in each group were counted from above experiments and shown as graph (n=3, mean ±S.D.). MCFC3 versus MCFC3Bcl-2-GFP cells and MCFC3 versus MCFC3Bcl-xL-GFP cells (P<0.001) for 12 h and (P<0.005) for 24 h treatment. Cyt. c release was checked by western blot in cytosolic fraction of MCFC3 vector, MCFC3 Bcl-2 GFP and MCFC3 Bcl-xL GFP-overexpressing cells after treatment with PAC-1 with different time points

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