Figure 2.

DAB2IP controls the intrinsic apoptosis of PCa cells following ADT. PCa sublines were cultured in Phenol Red-free RPMI-1640+5% CS-FBS for 3 days. (a) C4-2 (left) and LAPC-4 (right) sublines were then collected for Annexin V/FITC staining and flow cytometric analysis, and data (means±S.E.M.) were obtained from three independent experiments; *P<0.05. (b) Total lysates from C4-2 sublines were prepared to analyze cleaved PARP, Caspase-9 and cleaved Caspase-3 by western blotting; GAPDH was used as a total loading control. (c) C4-2 sublines were collected for JC-1 staining and flow cytometric analysis, and data (means±S.E.M.) were obtained from three independent experiments; *P<0.05. (d) Mitochondrial (left) or cytosolic (right) fractions from C4-2 sublines were prepared to analyze cytochrome c, Omi/HtrA2 and Smac by western blotting, COX IV and GAPDH were used as mitochondrial or cytosolic loading controls, respectively. (e) Cell lysates of C4-2 and LAPC-4 sublines were extracted for western blotting, to detect DAB2IP, survivin, Bcl-2, Bax, Bcl-xL and Mcl-1. Actin was used as loading control. (f) C4-2 and LAPC-4 sublines were transiently transfected with survivin pLuc-230 for 48 h along with the pRL-SV40 Renilla luciferase construct as internal control and then the promoter activity of survivin was determined by Dual-Luciferase Reporter Assay. Each result was performed in triplicate. *P<0.05