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Figure 1. Apoptosis in bak–/–/bax–/– cells induced by gossypol. A) bak–/–/bax–/– cells were treated with 20 µM gossypol for 24 h, followed by fixation and stained with Hoechst33342, and visualized by fluorescent microscopy. Arrows indicate the condensed, fragmented, brightly stained nuclei, which are the hallmark of apoptosis. Bar diagram shows the ratio of Hoechst positive cells. Data were the mean value of three independent experiments with each count of no less than 200 cells. *P < 0.05. B) Flow cytometric analysis of apoptosis in bak–/–/bax–/– cells following treatment with 20 µM gossypol for the indicated times. The percentage of apoptotic cells was determined by Annexin V/PI staining. C) bak–/–/bax–/– cells were treated with 20 µM gossypol for the indicated time or with indicated compound for 48 h, and the percentage of cells with activated caspases were characterized as those that stained with the FITC in situ marker. Data represent the mean values of three independent experiments. *P < 0.05. D) bak–/–/bax–/– cells were exposed to 20 µM gossypol in the presence of 100 µM zVAD-fmk or 5 mM autophagic inhibitor 3-MA for 24 h. The percentage of cell death was determined by Annexin V/PI staining followed by flow cytometric analysis. Results are expressed as the mean (±SD) for three independent experiments. *P < 0.05.
