Fig. 3

miR-146a-5p inhibits proliferation and promotes differentiation in C2C12 cells. (a) RT-qPCR to detect the expression of miR-146a-5p in proliferating C2C12 cells (n = 12). (b) The expression of miR-146a-5p gene in proliferating C2C12 cells after transfection with mimics and inhibitors (n = 12). (c, d) EdU image and statistical analyses of C2C12 (scale bar = 50 μm) (n = 12). (e) CCK-8 result of C2C12 (n = 8). (f, g) Cell cycle analysis of C2C12 by flow cytometry and statistical results (n = 3). (h) RT-qPCR analysis for Cyclin A2, Cyclin B1, Cyclin D1, PCNA and P21 in C2C12 (n = 6). (i, j) The protein levels of Cyclin A2, Cyclin D1, Cyclin E1, and PCNA by Western blot and the statistical analyses results in C2C12 (n = 3). (k) The expression of miR-146a-5p gene in differentiating C2C12 (n = 12). (l-n) Representative muscle fiber immunofluorescent MyHC staining in C2C12 (scale bar = 100 μm) (n = 4). (o) RT-qPCR analysis for MyoD, MyoG, Pax7, Fbx32, and MuFR in C2C12 (n = 9). (p-q) The protein levels of MyHC, MyoD, MyoG, Fbx32, and MuFR by Western blot and the statistical analyses results in C2C12 (n = 3). (r) RT-qPCR analysis for MyHC I, MyHC IIa, MyHC IIb and MyHC IIx in C2C12 (n = 9). (s) RT-qPCR analysis for IL-1β, IL-6 and TNF-α in C2C12 (n = 9). Values are presented as means ± SEM, *P <0.05, and **P <0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups