Fig. 3

Mechanism of promotion of C2C12 cell proliferation by MBO supernatant (MBOS). RNA sequencing analysis of C2C12 myoblasts cultured in serum-free control medium, growth medium (FBS), and MBOS (A). Heatmap of differentially expressed genes (DEGs) in C2C12 cells cultured in control, FBS, and MBOS (B). Genes were selected based on an adjusted P-value < 0.05, and hierarchical clustering revealed distinct transcriptional profiles across conditions. Volcano plot illustrating the DEGs between C2C12 cells cultured in serum-free control medium and MBOS (C). Genes with adjusted P-value < 0.05 and absolute log₂ fold change > 1 were considered significant. Gene Set Enrichment Analysis (GSEA) comparing transcriptomic profiles under control and MBOS conditions (D). Transcript-level expression of CCNB1 and CDK1 in MBOS-treated cells, as measured by FPKM (E, n = 3) and quantified using PCR (F). The expression level of each gene was quantified based on the ratio of the expression level to GAPDH and is shown as a fold increase relative to the control. The results are expressed as mean ± SEM (n = 4). *P < 0.05 vs. Control. The expression of CCNB1 and CDK1 in C2C12 cells after MBOS treatment (24 h) was determined using western blot analysis (G). The expression levels were quantified and plotted based on the ratio of the expression level to that of the control (G, n = 5). The results are expressed as mean ± SEM. Equal protein loading was confirmed using the VCP antibody as a control. ∗ P < 0.05 vs. Control.