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. 2015 Dec 8;291(5):2181–2195. doi: 10.1074/jbc.M115.676510

FIGURE 5.

FIGURE 5.

MEK-ERK1/2 activation is required for AA-induced C2C12 cell proliferation through up-regulation of cyclin D1 expression. a, C2C12 cells were treated with 5 mm AA for 24 h. Cell lysates were used for Western blot detection of cyclin D1, cyclin E, Cdk2, and Cdk4. β-Actin served as a loading control. PBS treatment served as control (Con). b, proteins in total lysates of C2C12 cells treated with different concentrations of AA (0.1, 0.5, 1, and 5 mm) and IGF1 (100 ng/ml) for 24 h were resolved by SDS-PAGE and analyzed by Western blotting for cyclin D1, Cdk2, and β-actin. c, efficiency of cyclin D1 knockdown in C2C12 cells was examined by Western blotting. d, cell proliferation was analyzed in cyclin D1-knockdown C2C12 cells treated with 5 mm AA or PBS for 24 h. e, expression levels of phospho (p)-MEK, total (t)-MEK, p-ERK, t-ERK, and β-actin proteins in C2C12 cells treated with 5 mm AA for different times (0.5, 1, 3, 6, and 12 h) were analyzed by Western blotting. f, C2C12 cells were pre-treated with the MEK inhibitor PD98059 (20 μm) for 1 h prior to AA (5 mm) treatment. The levels of cyclin D1, p-ERK, t-ERK, and β-actin were determined by Western blotting. g, proliferation of C2C12 cells treated with AA (5 mm) with or without PD98059 (20 μm) was analyzed by FACS. h, cyclin D1 expression was detected in the ERK1/2-knockdown C2C12 cells treated with 5 mm AA or PBS for 12 h. i, cell proliferation was analyzed in ERK1/2-knockdown C2C12 cells treated with 5 mm AA or PBS for 24 h. j, representative sections of AA-treated TA muscles, with and without PD98059 (20 μm) treatment, 2 days after injury immunostained for MyoD (green), and nuclei (DAPI; blue). Right panel shows merged image with DAPI. k, quantification of MyoD+ cells in sections described in j. l, expression of Pax7, cyclin D1, p-ERK, and t-ERK in damaged TA muscles treated with AA in the presence or absence of PD98059 was analyzed by Western blotting. Scale bar, 50 μm. Similar results were obtained in three separate experiments. Data are presented as means ± S.E. (error bars; *, p < 0.05). NS, not significant.