FIGURE 4.

Bcl-2 knockdown-induced apoptosis in Jurkat cells can be completely inhibited by Bak knockdown but only partially inhibited by Bim and/or Puma knockdown. A, 48 h after transfection with pCMS5A (empty vector, which encodes EGFP-histone H2B), pCMS5A/Bcl-2 shRNA 1, pCMS5A/Bcl-2 shRNA 2, or pCMS5A/Bcl-2 shRNA #1/Bcl-2* (shRNA-resistant Bcl-2), Jurkat cells were collected and stained with APC-annexin V. The percentage of EGFP⁺ cells that stained with APC-annexin V is indicated. Cells were also subjected to immunoblotting. B and C, 48 h after transfection with pCMS5A or pCMS5A/Bcl-2 shRNA, Jurkat cells were collected, fixed, and stained with Ab-1, which recognizes active Bak. A representative experiment (B) and summary of percentage of EGFP⁺ cells stained with Ab-1 (C) is shown. D, 48 h after co-transfection of control siRNA, Bak siRNA, or Bax siRNA with EBFP-histone H2B, Jurkat cells were collected and transfected with pCMS5A vector or pCMS5A/Bcl-2 shRNA for another 48 h and then stained with APC-annexin V. The percentage of EBFP⁺EGFP⁺ cells that stained with annexin V is indicated. Cell lysates were probed to confirm knockdown. E, 48 h after co-transfection of control siRNA, Bim siRNA, Puma siRNA, or Bim siRNA + Puma siRNA with EBFP-histone H2B, Jurkat cells were collected and transfected with pCMS5A or pCMS5A/Bcl-2 shRNA for another 48 h and then collected and stained with APC-annexin V. The percentage of EBFP⁺EGFP⁺ cells that stained with annexin V is indicated. Cell lysates were probed to confirm knockdown.