Figure 1. DNA Damage–Induced Bcl-x_L Deamidation Is Mitochondrial Apoptosis–Independent.

(A) Wild-type thymocytes were pre-incubated with or without Z-VAD-fmk (200 μM), and were then cultured with or without etoposide for 24 h, harvested, and apoptosis was measured by measuring the sub-G1 peak by flow cytometry. The histograms (right panel) represent mean values ± SD (n = 3).

(B) Aliquots of the cells from (A) incubated in the presence or absence of Z-VAD-fmk (200 μM) were analysed for the expression of Bcl-x_L and tubulin (as loading control) by immunoblotting. The upper and lower bands of Bcl-x_L were quantified and expressed as a percentage of total Bcl-x_L. The percentages shown below each lane are means ± SD (n = 3).

(C) Plasmids of shRNA Bax (GFP) and shRNA Bak (DsRed) were cotransfected into purified DN thymocytes using an Amaxa nucleofactor kit. 48 h later, GFP⁺ DsRed⁺ cells were purified by flow cytometry and treated with etoposide (Etop, 25 μM) for 30 h or exposed to irradiation (IR, 5 Gy) followed by 30 h in culture. DN thymocytes transfected with negative control plasmids were treated in parallel. Cells were then processed for immunoblotting with Bcl-x_L antibody. The immunoblot was reprobed for Bax and Bak to check the efficiency of gene knockdown. Tubulin was also reprobed as a loading control.