Ngăn ngừa quá trình Tự Hủy Tế Bào bởi Bcl-2: Ngăn chặn Sự Giải Phóng Cytochrome c khỏi Ty thể

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HL-60 cells overexpressing Bcl-2 and the vector control cells were harvested by centrifugation at 600 g for 10 min at 4°C. The cell pellets were washed once with ice-cold phosphate-buffered saline (PBS) and resuspended with five volumes of buffer A (20 mM Hepes-KOH pH 7.5 10 mM KCl 1.5 mM MgCl 2 1 mM sodium EDTA 1 mM sodium EGTA 1 mM dithiothreitol and 0.1 mM phenylmethylsulfonyl fluoride) containing 250 mM sucrose. The cells were homogenized with 10 strokes of a Teflon homogenizer and the homogenates were centrifuged twice at 750 g for 10 min at 4°C. The supernatants were centrifuged at 10 000 g for 15 min at 4°C and the resulting mitochondria pellets were resuspended in buffer A containing 250 mM sucrose and frozen in multiple samples at −80°C. The supernatants of the 10 000 g spin were further centrifuged at 100 000 g for 1 hour at 4°C and the resulting supernatants (designated S-100) were divided into samples and frozen at −80°C for further experiments.

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neo and Bcl-2 cells were set up and treated with staurosporine as described in (17). After centrifugation at 600 g for 10 min at 4°C the cell pellets were resuspended in PBS containing 5 μM rhodamine 123 (Sigma) and incubated at 37°C for 15 min. The cell suspension was centrifuged in a microcentrifuge for 30 s and the pellet was resuspended in 20 μl of PBS plated onto a 25-mm round glass cover slip coated with poly-D-lysine and mounted into a perfusion chamber for confocal imaging. The fluorescence images were collected with a Meridian Insight-Point Laser scanning confocal microscope (Meridian Instrument) equipped with a Zeiss Axioplan microscope. The objective lens was a 100× numerical aperture 1.4 PlanApo lens. The aperture size of the pinhole was 10 to 40 μm. Confocal optical sections were estimated to be less than 1 μm in thickness. Cells were excited with the 488-nm line of an argon laser and emitted fluorescence was detected through a 530/30 band-pass filter with an intensified cooled charge-coupled-device camera. A typical cell from a population of ∼100 was presented.

We thank R. Jemmerson of University of Minnesota for monoclonal antibody to cytochrome c; S. McKnight J. L. Goldstein and M. S. Brown for critically reading the manuscript; and T. Greenamyer for help with confocal microscopy. Supported by the start-up fund from Emory University an American Cancer Society research grant and a Junior Faculty award (to X.W.).