Figure 6

The role of ASK1 in MC3-mediated cellular apoptosis. (A) MC3 interrupted the binding between ASK1 and Trx. Panc1 cells were incubated with 5 μM MC3 as indicated. The cells were collected for immunoblot detected by Trx and ASK1 antibodies and for immunoprecipitation precipitated with ASK1 antibody and then immunoblotted with ASK1 and Trx antibodies. (B) ASK1 siRNA inhibited phophorylation of p38-MAPK. HeLa cells were transfected with ASK1 siRNAs for 48 h. The cells were treated with 10 μM MC3 for 1 h and collected for immunoblot. No target siRNA was used in the transfection as negative control. (C) Resistance to MC3 in ASK1-deficient HeLa cells. After 48 h transfection with ASK1 siRNAs HeLa cells were incubated with 5 μM MC3 for 24 h and the apoptotic cells were analyzed by FACS staining with annexin v/PI. (D) 50 nM of ASK1 siRNA was sufficient to knockdown endogenous ASK1 in ASPC1 (left) and Panc1 (right) after 48 h detected by immunoblotting. (E) ASK1 siRNA rescued MC3-induced cell apoptosis in annexin v/PI assay in Panc1. (F) ROS scavengers, p38i and ASK1 siRNA attenuated cellular damage upon MC3 in ASPC1 after 24 h treatment. (G) Schematic model of MC3 induced cell apoptosis in pancreas. The densitometric analyses of corresponding immunoblots are provided in supporting information.