Fig. 4

Akt confers CDDP resistance by suppressing Oma1 cleavage, L-Opa1 processing, mitochondrial fragmentation and apoptosis. A HA-DN-Akt treatment enhanced CDDP-induced p-p53 (ser15), Oma1 (40 KDa) and L-Opa1 processing [**p < 0.01, ***p < 0.001, (versus CDDP in absence of DN-Akt); n = 3]. C13* (chemoresistant CECA cells) were forced expressed with HA-DN-Akt or Lac Z (Multiplicity of Infection (MOI) = 0–80, 24 h) and treated with CDDP (0–10 μM, 24 h). The contents of HA tag, p-p53 (ser15), Phb1,Oma1, Opa1, and GAPDH (loading control) were determined by WB. (B) HA-DN-Akt treatment sensitized C13 cells to CDDP-induced apoptosis [***p < 0.001 (versus CDDP in absence of DN-Akt); n = 3]. C13* cells were treated with HA-DN-Akt or Lac Z (MOI = 0–80, 24 h), and then with CDDP (0–10 μM, 6 h). Apoptosis was examined by Hoechst assay. C Akt down-regulation sensitized C13 cells to CDDP-induced mitochondrial fragmentation [***p < 0.001 (versus CDDP in absence of DN-Akt); n = 3]. Mitochondrial phenotypes were examined by immunofluorescence confocal microscopy. D Akt conferred resistance in C13 cells by suppressing CDDP-induced the interaction of p-p53 (ser15) with Phb1 and Bak. C13* cells were treated with HA-DN-Akt or Lac Z (MOI = 0 or 80, 24 h), and then with CDDP (0–10 μM, 6 h). Contents of Bak, Phb1, p-p53 (ser15), and GAPDH were examined by WB. Cell lysates were immunoprecipitated with IgG (control; lanes 1) or Bak antibody. Protein-protein interaction was determined by IP-Western. Bak immunoprecipitates were immunoblotted [IP: anti-Bak, WB: anti-Bak, Phb1, p-p53 (ser15)]. Results are expressed as mean ± SEM (n = 3) and analyzed by 2-way ANOVA and Bonferroni post-hoc test. [***p < 0.001 versus CDDP in absence of DN-Akt); n = 3]