Membrane Bound Monomer of Staphylococcal α-Hemolysin Induces Caspase Activation and Apoptotic Cell Death despite Initiation of Membrane Repair Pathway
Figure 6
H35N induced cell death and changes in nuclear morphology in presence of pan-caspase inhibitor zVADfmk:
(A) A431 cells either left untreated or treated with H35N (8 µg/ml) or zVADfmk (50 µM) for 2 hr followed by addition of H35N (8 µg/ml) for the indicated time which were then mildly trypsinised and viable cells were counted after staining with trypan blue as mentioned in materials and methods. Percentage of stained cells were plotted. The graph represents one of the two independent experiments. (B–D) Cells were treated as mentioned above, stained with PI (2 µg/ml) for 5 min. The samples were analyzed by flow cytometry and atleast 10,000 events were acquired for each sample. (B) control (C) H35N treated cells (D) H35N+ zVADfmk treatment. Apoptotic cell morphology was detected by decrease in forward scatter. (E) Cells post treatment for the following time points were fixed and stained with Hoechst 33342 as mentioned in materials and methods. The lower panel shows merged image of Hoechst staining with phase contrast.