IL-13Rα2 Regulates C2C12 Myoblast Proliferation via the Akt–Cyclin D1–CDK4 Pathway

Figures

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  Figure 1. Effects of IL-13Rα2 knockdown on myogenic differentiation and myotube morphology in C2C12 cells. (A) Representative Western blot analysis of IL-13Rα2 protein expression. GM and DM3 indicate cells cultured in growth medium and differentiation medium for 3 days, respectively. KD indicates IL-13Rα2 siRNA-transfected myoblasts cultured in growth medium. The marked band corresponds to IL-13Rα2. (B) Relative IL-13Rα2 mRNA expression measured by quantitative PCR in control siRNA-transfected (Ctrl) and IL-13Rα2 siRNA-transfected (KD) cells. n = 4 per group. (C) Representative immunofluorescence images of Myosin Heavy Chain (MyHC; red) in differentiated myotubes. Nuclei were counterstained with DAPI (blue). Scale bars: 50 μm. (D) Quantification of the number of nuclei per myotube in the Ctrl and KD groups. n = 4 per group. (E) Differentiation index, calculated as the percentage of nuclei located within MyHC-positive myotubes. n = 4 per group. (F) Quantification of myotube diameter. n = 4 per group. (G) mRNA expression of myogenic differentiation- and fusion-related genes (MyoG, Myomaker, Myomerger, and MyHC4), as measured by qRT-PCR. n = 6 per group. Data are presented as mean ± SD.…

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  Figure 2. IL-13Rα2 knockdown enhances proliferation and alters Akt and ERK phosphorylation in C2C12 myoblasts. (A) Representative BrdU immunofluorescence images of Ctrl and KD cells cultured in the growth medium (GM). Nuclei were stained with DAPI (blue). Scale bars: 50 μm. (B) Quantification of BrdU-positive cells (green) showing significantly increased proliferation in KD cells. n = 6 per group. (C) Representative Western blots showing total and phosphorylated forms of Akt, ERK1/2, and p38. GAPDH was used as the loading control. (D) Quantification of pAkt, pERK1/2, and pp38 levels, as the ratio to their respective total protein levels. n = 6 per group. (E) qRT-PCR analysis of Cyclin D1 and CDK4 mRNA expression. n = 6 per group. (F–H) Representative immunofluorescence images and quantification of Cyclin D1-positive and CDK4-positive cells (green) in the Ctrl and KD groups. n = 6 per group. Scale bars: 50 μm. Data are presented as mean ± SD. Statistical significance was determined using an unpaired Student’s t-test. * p < 0.05.

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  Figure 3. Akt inhibition abolishes the enhanced proliferation and Cyclin D1/CDK4 upregulation caused by IL-13Rα2 knockdown. (A) Representative BrdU staining of Ctrl, KD, and KD cells treated with an Akt inhibitor (10 μM MK-2206). Scale bar = 50 μm. (B) Quantification of BrdU-positive cells (green). The promotion of proliferation due to KD was reversed by Akt inhibition. n = 6 per group. (C) Representative Western blots of Cyclin D1 and CDK4 in Ctrl, KD, and KD + Akt inhibitor groups. (D,E) Quantification of Cyclin D1 and CDK4 protein levels, showing that Akt inhibition suppresses KD-induced upregulation. n = 6 per group. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA with Tukey–Kramer post hoc test for multiple comparisons. * p < 0.05.

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  Figure 4. ERK activation does not modify the proliferative phenotype of IL-13Rα2 knockdown myoblasts. (A) Representative BrdU staining of Ctrl, KD, and KD cells treated with the ERK activator (rel)-AR234960 (10 μM). Scale bar = 50 μm. (B) Quantification of BrdU-positive cell (green) numbers showing no significant difference between KD and KD + ERK activator groups. n = 6 per group. (C) Representative Western blots of Cyclin D1 and CDK4. (D,E) Quantification showing elevated Cyclin D1/CDK4 protein levels in KD cells relative to Ctrl, with no further increase after ERK activation. n = 5 per group. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA with Tukey–Kramer post hoc test for multiple comparisons. * p < 0.05.