Effects of FRNK overexpression on LN-mediated Gem chemoresistance. Parental AsPC-1 cells and vector-transfected (Vector) and pcDNA3.1-FRNK-transfected (Pool) clones were treated with Gem (0.1 μM) for 72 h. Cell apoptosis was examined by Hoechst staining (the arrow indicates the apoptotic cells) (A), flow cytometry analysis of Annexin-V labeling(B) and western blot analysis of cleaved caspase-3 protein expression(C). Bars represent the mean of three independent experiments ± SE. *, P < 0.05, vs. parental cells on plastic; #, P < 0.05, vs. parental cells or vector cells on LN. D, After plated on LN for 24 h, western blot analysis was used to detect the expression of Bad, p-Bad (pS136), p-Bad (pS112), Bcl-2, Bax and survivin in parental AsPC-1 cells and vector and pool clones on LN. Parental cells on plastic were used as control. The membranes were probed with anti-β-actin antibody to ensure even loading of proteins in each lane.