Fig. 5.

AOPPs-induced G1 phase arrest in IEC-6 cells via the activation of JNK. IEC-6 cells were treated with 200 μg/ml of AOPPs for the time periods indicated (a) or pretreated with a JNK inhibitor (SP600125), at 10 μM, for 1 h and then incubated with 200 μg/ml of AOPPs for 24 h (b–e). (a) Representative Western blotting and densitometry quantification showing that AOPPs enhanced the phosphorylation of JNK in IEC-6 cells in a time-dependent manner. (b) Flow cytometry showing that the JNK inhibitor suppressed AOPPs-induced G1 phase arrest in IEC-6 cells. Quantitative analyses of FACS data represent G1 phase cell percentages. (c) Immunofluorescence staining for cyclin E, CDK2 and p27kip1 revealed that a JNK inhibitor enhanced the expression of cyclin E and CDK2 and weakened the expression of p27kip1 AOPPs-treated cells. Scale bar, 20 μm. (d) Representative Western blotting and bar graphs depicting densitometry quantification revealed that the AOPPs-induced reduction of cyclin E and CDK2 and upregulation of p27kip1 could all be blocked by a JNK inhibitor. (e) Bar graph showing the results of qPCR assays for mRNA expression. The JNK inhibitor increased cyclin E and CDK2 expression and decreased p27kip1 expression in IEC-6 cells after AOPPs treatment. Relative protein and mRNA levels were normalized with GAPDH, respectively. (f) Results of co-immunoprecipitation analysis showing that the JNK inhibitor attenuated the AOPPs-induced increased binding of p27kip1 to cyclin E-CDK2 complexes in IEC-6 cells. Data are presented as mean ± SD from three independent experiments; error bars indicate SD. *P < 0.05 and **P < 0.01 versus control; #P < 0.05 and ##P < 0.01 versus AOPPs-treated cells. JNK, c-jun N-terminal kinase. +SP600125, AOPPs plus an inhibitor of JNK treatment.