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. 2024 Oct 4;12:RP86827. doi: 10.7554/eLife.86827

Figure 4. The MSTNDel73C mutation with FGF5 knockout contributes to muscle phenotype via MEK-ERK-FOSL1 axis.

(A) Go enrichment analysis of DEGs. Among them, the top 20 entries with significant enrichment are listed in biological process (BP). CC, cellular component; MF, molecular function. (B) KEGG enrichment analysis of DEGs. (C) The mRNA expression level of FOSL1 both at gluteus medius and longissimus dorsi in WT (n=3) and MF+/- (n=4) sheep. (D) The mRNA expression level of FOSL1 both at GM and DM2 in WT and MF+/- cells (n=3). (E) The expression level of FOSL1 mRNA during myogenic differentiation (n=3). (F) The protein-protein interaction (PPI) analysis of FOSL1, c-Fos and MyoD1. (G) The mRNA expression level of c-Fos and MyoD1 at GM in WT and MF+/- myoblasts (n=3). (H) Schematic diagram of MyoD1 gene body, promoter region and binding sites. (I–J) FOSL1 recognition motif in the MyoD1 promoter region. (K) FOSL1 ChIP-qPCR of motif 1 recognition region (n=3). (L) FOSL1 ChIP-qPCR of motif 2 recognition region (n=3). (M) Dual luciferase assay for the effect of FOSL1 on MyoD1 promoter activity (n=4). (N) Western blot of FOSL1, c-Fos, and key kinases of MAPK signaling pathways at GM. (O) Quantification of protein expression of FOSL1, c-Fos, and key kinases of MAPK signaling pathways at GM (n=3). (P) Western blot of FOSL1, c-Fos, and key kinases of MAPK signaling pathways at DM2. (Q) Quantification of protein expression of FOSL1, c-Fos, and key kinases of MAPK signaling pathways at DM2 (n=3). Data: mean ± SEM. Unpaired student’s t-test was used for statistical analysis. All student’s t-test were performed after the equal variance test, otherwise the t-test with Welch’s correction were used. *p<0.05, **p<0.01, and ***p<0.001.

Figure 4—source data 1. Uncropped and labeled blots for Figure 4N and Figure 4P.
Figure 4—source data 2. Raw unedited blots for Figure 4N and Figure 4P.

Figure 4.

Figure 4—figure supplement 1. The effects of MSTN signaling pathway and MF+/- serum on proliferation and differentiation of skeletal muscle satellite cells.

Figure 4—figure supplement 1.

(A) The mRNA expression levels of type I and type II receptors of MSTN between WT (n=3) and MF+/- (n=4) sheep gluteus medius. (B) The mRNA expression levels of the Smad family downstream of MSTN between WT (n=3) and MF+/- (n=4) sheep gluteus medius. (C) The mRNA expression levels of the Jun family downstream of MSTN between WT (n=3) and MF+/- (n=4) sheep gluteus medius. (D) The number of cells detected by CCK-8 at 0 hr, 24 hr, and 48 hr after culturing cells with serum from WT sheep and MF+/- sheep (n=4–5). (E) The mRNA expression levels of FOSL1 and myogenic differentiation markers between WT and MF+/- sheep skeletal muscle satellite cells cultured and induced differentiation in sheep serum (n=4). (F) The mRNA expression levels of FOSL1 and myogenic differentiation markers in skeletal muscle satellite cells cultured and induced differentiation by serum from WT and MF+/- sheep (n=4). Data: mean ± SEM. Unpaired student’s t-test and chi square test were used for statistical analysis. All student’s t-test were performed after the equal variance test, otherwise the t-test with Welch’s correction were used. *p<0.05, **p<0.01, and ***p<0.001.