Figure 5.

CTD promoted mitochondrial localization of Nur77 and subsequent Bcl-2 transformation. (A) HL-60 cells were treated with CTD as indicated for 48 h and then subjected to Western blotting for the detection of Nur77 protein level. (B) Western blotting detection of the expression of Nur77 in HL-60^shNur77 cells and HL-60^ShNC cells. (C) HL-60^shNur77 cells and HL-60^pLKO.1 cells were treated with or without 4 μM CTD for 48 h, respectively. Apoptotic cells were determined by flow cytometry (n = 3). (D) After treatment of HL-60 cells with or without 4 μM CTD for 24 h, cell lysates were subjected to Western blotting for analyzing Nur77 and HSP60 in mitochondria (M) and Nur77 and α-tubulin in cytoplasm (C). (E) HL-60 cells were treated with or without 4 μM CTD for 24 h. Cell lysates were immunoprecipitated with anti-Nur77 or anti-Bcl2 antibody, and then Nur77 and Bcl-2 were detected by Western blotting. Input, cell lysates without IP process is set as a positive control. IgG, IP with anti-immunoglobulin G (IgG) is set as a negative control. The blots are a representative of three independent experiments. (F) HL-60 cells were treated with CTD for 24 h and then Bcl2 BH3 Domain Exposure was determined by flow cytometry with an antibody against Bcl-2 (BH3) (n = 3).