Figure 2.

The MAPK signaling pathway is critical for the promotion of C2C12 cell proliferation by Msx1. (A) Heatmap of RNA-seq data from C2C12 cells overexpressing Msx1 or the control based on the log₂ intensity (n = 3). (B) KEGG signaling pathway enrichment of DEGs in C2C12 cells overexpressing Msx1 or the control, ranked by the P value. (C) GSEA result of RNA-seq data of C2C12 cells overexpressing Msx1 and the control. The MAPK signaling pathway is enriched with FDR of 0.242. (D) Western blotting assays to detect the level of p-Erk1/2 in Msx1-overexpressing and the control C2C12 cells. (E) Western blotting assays to detect the level of p-Erk1/2 in Msx1-KO and the control C2C12 cells. Numbers under western blot bands represent relative quantifications over t-Erk1/2 quantified by ImageJ software. Statistical analysis of western blot grey scale values is shown in (F) (n = 3, wild-type was set as 1). Values are the means ± SD. **P < 0.001. (G) Flow cytometry assays to evaluate the impact of PD0325901 on C2C12 cell cycle progression promoted by Msx1. C2C12 cells overexpressing Msx1 or the control were treated with 1 μM PD0325901 or DMSO for 24 h, respectively. (H) Statistical analysis of the cell cycle of differently treated C2C12 cells in (G). Values are the means ± SD. **P < 0.001, *P < 0.01. (I) CCK-8 assays to assess the impact of PD0325901 on C2C12 cell proliferation promoted by Msx1. Values are the means ± SD. ***P < 0.0001, **P < 0.001. (J) Western blotting assays to detect the impact of PD0325901 on the expression of a proliferation marker promoted by Msx1.