FIG. 2.

Effect of capsaicin on ASK1 cascade in cells overexpressing ASK1 or Trx. (A) AsPC-1 cells were treated with dimethyl sulfoxide (DMSO) or 150 μM of capsaicin, immunostained with p-ASK1 Thr845 (red) and actin (green) antibodies, and visualized under a fluorescence microscope (Olympus Inc.). AsPC-1 cells were transiently transfected with 2 μg of (B) hemagglutinin (HA)-tagged WT-ASK1 or (C) Flag-tagged WT-Trx using the FuGENE transfection reagent for 24 h, and the transfected cells were treated with DMSO or 150 μM capsaicin for 24 h. The expressions of Trx, p-ASK1 (Thr845), and ASK1 and cleavage of caspase-3 were evaluated by immunoblotting. Each blot was stripped and reprobed with antiactin antibody to ensure equal protein loading. Bar diagram shows the quantitation of respective western blots normalized with actin. (D) Apoptosis was determined using annexin-V/fluorescein isothiocyanate (FITC) and propidium iodide in AsPC-1 cells transfected with HA-tagged WT-ASK1 or Flag-tagged Trx as described above and then treated with DMSO or 150 μM of capsaicin for 24 h. Western blot experiments were performed three times independently with similar observations made in each experiment. Values are means±SD of three independent experiments. *Statistically different compared with control (p<0.05), **statistically different when compared with capsaicin treatment (p<0.05), and ***statistically different when compared with capsaicin treatment and WT-ASK1 transfection (p<0.05), as analyzed by the one-way analysis of variance (ANOVA) followed by the Bonferroni post-hoc test. (To see this illustration in color the reader is referred to the web version of this article at www.liebertpub.com/ars).