Figure 4. The effect of MAPK15 knockdown on cell cycle and c-Jun stability.

The SNU-601 cells were transfected with MAPK15 siRNA (siMAPK15) or nonspecific siRNA (siCtrl). A. The mRNA and protein levels of MAPK15 were measured using qRT-PCR and immunoblot analysis, respectively, on the 3^rd day after siRNA transfection. Error bars indicate standard deviation (n = 3, *P < 0.05). B. Cell proliferation was detected by MTS assay. Absorbance at 490 nm was measured on the 1^st, 3^rd, and 5^th day after siRNA transfection. Error bars indicate standard deviation (n = 4, *P < 0.05). C. On the 3^rd day of post-siRNA transfection, cells were treated with 10 μM BrdU for 2 hours, and collected. The cells were incubated with a FITC-conjugated anti-BrdU antibody. Total DNA was stained with 7-AAD. D. The protein levels of c-Jun and P-c-Jun were detected by immunoblot analysis on the 3^rd day after siRNA transfection. MAPK15 knockdown experiment was performed three times, and the ratio of phosphor-c-Jun to total c-Jun was found to be significantly decreased in cells transfected with siMAPK15 than in those transfected with siCtrl (P = 0.02, Wilcoxon rank-sum test). E. The mRNA levels of c-Jun were detected by qPCR on the 3^rd day after siRNA transfection. F. On the 3^rd day after siRNA transfection, cells were treated with cycloheximide (80μg/ml), a protein synthesis inhibitor, for 0, 1, 2, or 4 hours, and the c-Jun protein level was analyzed by immunoblotting. The experiment was performed twice, and a similar result was obtained.