Id4 promotes apoptosis by regulating mitochondrial membrane potential and the expression of pro-apoptotic genes. A: percent cells undergoing apoptosis was determined by propidium iodide and Annexin V staining followed by flow cytometery. Significant increase in apoptosis (***: P < 0.001) was observed in DU145 cells over-expressing Id4 (D + Id4) when compared with DU145 cells alone (D). A significant decrease in apoptosis was observed in LNCaP cells that lacked Id4 (L-Id4) as compared to LNCaP cells (L, ***: P < 0.001). B. Percent cells with high mitochondrial membrane potential (Gated, FL2 > 100 fluorescence units). In the presence of Id4 (D + Id4 and L), the mitochondrial membrane potential decreased as compared to the corresponding cells that lack Id4 (D and L-Id4). (***: P < 0.001–L vs. L-Id4 and D vs. D + Id4). C. Western blot analysis of p21, BAX, conformation specific BAX (BAX6A7) and PUMA in D, D + Id4, L and L-Id4 cells. GAPDH was used as loading control. Representative western blots of three different experiments are shown. D. Real time quantitative analysis of p21, BAX and PUMA expression in D, D + Id4, L and L-Id4 cells. The mean ± SEM of three experiments in triplicate is shown. The ΔΔCt (normalized to GAPDH) between D and D + Id4 (D normalized to 1, designated as “a”) and between L and L-Id4 (L normalized to 1, designated as “b”) is shown (*: P < 0.001). E. Immuno-cytochemical analysis demonstrating co-localization of conformation specific BAX (using BAX 6A7 antibody) with mitochondrial pyruvate dehydrogenase (PDH). Blue: DAPI, red: PDH, green: BAX 6A7 and yellow: co-localization of BAX and PDH (observed only in LNCaP and DU145 + Id4 panels. Representative images from three different experiments are shown.