Fig. 3.

The effect of pharmacological inhibition of JNK on FOXO1 activation in cultured gastric cancer cells. (a-c). SNU-638 cells were cultured without treatment (Ctrl) or treated with DMSO vehicle control (DMSO) or SP600125 (SP) for 24 h. a Cells were treated with various concentrations of SP. Protein expressions were analyzed by Western blotting using specific antibodies against pJNK^{Thr183/Tyr185} (pJNK), cyclin D1, pFOXO1^Ser256, total FOXO1, and β-actin. b Cells were cultured in the presence or absence of SP (20 μM) and FOXO1 transcriptional activity was determined by luciferase reporter assay, which was normalized by β-galatosidase activity. Luciferase activity in Ctrl was arbitrarily set to 1 and the activities of other cells were adjusted accordingly. Each bar represents the mean ± standard deviation. * P < 0.05 compared with untreated control groups (Ctrl and DMSO). c The effect of JNK inhibition on the subcellular localization of FOXO1 was observed by double immunofluorescence staining for pJNK (green) and FOXO1 (red). 4’,6-diamidino-2-phenylindole (DAPI) staining was performed for nuclear localization (blue). d Cells were transfected with non-targeting shRNA (shCtrl) or FOXO1 shRNA (shFOXO1). The effects of FOXO1 silencing on the expressions of total FOXO1, cyclin D1, pJNK, and total JNK were evaluated by Western blotting