Fig 1: The effect of MAPK15 knockdown on cell cycle and c-Jun stabilityThe 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 3rd 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 1st, 3rd, and 5th day after siRNA transfection. Error bars indicate standard deviation (n = 4, *P < 0.05). C. On the 3rd 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 3rd 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 3rd day after siRNA transfection. F. On the 3rd 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.
Fig 2: Multiplex ligation-dependent probe amplification (MLPA) of MAPK15Upper panel shows a representative image of capillary electrophoresis signals analyzed by MLPA and GeneMaker 2.0.0. Lower panel indicates DNA copy number of MAPK15 detected by MLPA in 6 paired samples of gastric cancer matched with the normal (268-1, 271-1, 272-2, 301-1, 685-1 and 685-2). Probe ratios below 0.7 and above 1.3 indicate loss and gain, respectively. The “T” and “N” represent tumor and normal tissues, respectively.
Fig 3: Immunohistochemical staining of MAPK15A. Anti-MAPK15 antibody was tested using immunofluorescence on control, SNU-601 cells, and on AGS cells transfected with MAPK15-pCMV6-Myc-DDK. Cultured cells were stained with anti-MAPK15 antibody. Alexa Fluor-488 goat anti-rabbit secondary antibody was used for fluorescence labeling of MAPK15. DNA was stained with DAPI. B. Protein levels of MAPK15 in concurrent legions (normal, adenoma, and carcinoma) from 45 gastric cancer patients were analyzed by immunohistochemistry. The MAPK15 is weakly stained in the cytoplasm. (X200). C. The prevalence of MAPK15 overexpression was calculated in normal, adenoma, and carcinoma legions from 45 patients. One patient did not have normal lesion, and some of patients did not have adenoma or carcinoma lesions, and therefore the total number of adenoma and carcinoma lesions was not 45. D. The expression status of MAPK15 was compared in concurrent legions individually, to assess the effect of MAPK15 overexpression on malignant transformation of the stomach. The overexpression of MAPK15 was found in 16 patients, and seven (44%) of the patients have MAPK15 overexpression in concurrent adenoma and carcinoma lesions, and 7 (44%) have MAPK15 overexpression only in carcinoma lesions.
Fig 4: DNA copy number alterations (CNAs) on chromosome 8A. The CNAs in 30 gastric cancers were analysed by agilent aCGH-400K and 10 by aCGH-244K. The diagram shows CNAs on chromosome 8 detected by aCGH-400K (left) and aCGH-244K (right). Vertical lines represent cytoband of chromosome 8. The red and green colors indicate regions of DNA copy number gains and losses, respectively. Arrows indicate samples with copy number gains of MAPK15. B. Aberrations around MAPK15 gene at 8q24.3 are shown. Vertical lines indicate log2-based intensity ratios values, and each colored horizontal line represents a copy number alteration. Log2 ratios of signal intensities of samples with normal copy number are plotted with the horizontal central line equal to zero. Horizontal lines above the 0 of log2-based intensity ratio in the aCGH-400K and aCGH-244K indicate samples with 8q24.3 amplification. Five horizontal lines above the zero in aCGH-400K and three horizontal lines in aCGH-244K indicate samples with copy number gains.
Fig 5: Effect of MAPK15 overexpression on c-Jun phosphorylationAGS cells were transfected with the pCMV6-Entry plasmid that express Myc-DDK tagged MAPK15 protein (MAPK15-Ove) or the Mock plasmid (Ctrl). A. MAPK15 protein levels were measured by immunoblot analysis on the 3rd day post-transfection. B. Cell proliferation was detected by MTS assay. Absorbance at 490 nm was measured on the 1st, 3rd, and 5th day post-transfection. Error bars indicate standard deviation (n = 4, *P < 0.05). C. The protein levels of c-Jun and P-c-Jun were detected by immunoblot analysis on the 3rd day post-transfection. D. AGS cells were treated with cycloheximide (80 μg/ml) for 0, 1, 2, or 4 hours, and the c-Jun protein level was measured by immunoblot analysis. The experiment was performed twice, and a similar result was obtained. E. c-Jun and phosphorylated-c-Jun levels were analyzed by western blotting after treating AGS cells with SP600125, U0126, or SB203580 as described in the Materials and Methods.
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