FIGURE 4.

ATAP does not require either Bax or Bak activity to induce apoptosis although BH3 peptides do. A, detection of Bax and Bak genes by PCR. 100 ng of genomic DNA isolated from cultured BMK-W2 (bax^+/+, bak^+/+) and BMK-D3 (bax^−/−, bak^−/−) cells was analyzed by PCR using primers designed to amplify an exon sequence of 149 bp in Bax gene and of 181 bp in Bak gene. PCR products were then analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining. B, viability of BMK W2 or BMK D3 cells transfected with 1 μg of GFP-ATAP or GFP-BH3 expression plasmid. At 24 h after transfection, cells were stained with DAPI and observed under a fluorescence microscope. Representative photographs show GFP-positive cells after 24 h of transfection. Bar, 10 μm. C, percentage of surviving cells was determined by the ratio of GFP-positive cells without DAPI staining to total GFP-positive cells. About 200 cells from three different fields were scored. Data are expressed as the means ± S.E. D, dose effect of GFP-ATAP and GFP-Bak BH3 on apoptosis of BMK D3 cells. Cells were transiently transfected with the indicated amounts of GFP-ATAP or GFP-Bak BH3 construct. Cell survival was measured by DAPI exclusion assay. Data are expressed as the means ± S.E. E, time course study of apoptosis induced by GFP-ATAP and GFP-Bak BH3 in BMK D3 cells. Cells were transiently transfected with 1 μg of GFP-ATAP or GFP-Bak BH3 construct. Cell survival was measured by DAPI exclusion assay. Data are expressed as the means ± S.E.