PFN1 Inhibits Myogenesis of Bovine Myoblast Cells via Cdc42-PAK/JNK

Figure 1. The effect of PFN1 on the differentiation of bovine skeletal muscle satellite cells: (A) qRT-PCR detection of mRNA expression level of differentiation marker genes in si-PFN1 or si-NC transfected DM2 myoblasts; (B) qRT-PCR detection of the mRNA expression level of differentiation marker genes in pcDNA-PFN1 or pcDNA3.1(+) transfected DM2 myoblasts; (C) Western blot and quantitative analysis of differentiation marker protein levels in si-PFN1 or si-NC transfected DM2 myoblasts; (D) Western blot and quantitative analysis of differentiation marker protein levels in pcDNA-PFN1 or pcDNA3.1(+) transfected DM2 myoblasts; (E) Myotube formation status in si-PFN1 or si-NC transfected DM2 myoblasts; (F) Myotube formation status during differentiation in pcDNA-PFN1 or pcDNA3.1(+) transfected DM2 myoblasts. si-NC stands for negative control, and pcDNA3.1(+) stands for empty vector control negative control. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 2. Identification of PFN1-interacting proteins: (A) identified proteins by CoIP-MS; (B–E) GO and KEGG analysis of PFN1 binding proteins; (F) protein-protein interaction analysis of PFN1 candidate binding proteins by STRING; (G) validation the binding of PFN1 and Cdc42 by Co-IP in DM2 myoblasts.

Figure 3. Regulation of Cdc42 by PFN1 and the effect of Cdc42 on the differentiation of bovine myoblasts: (A) qRT-PCR detecting the effect of interfering or overexpression of PFN1 on Cdc42 mRNA level in DM2 myoblasts; (B) Western blot detecting the effect of interfering with PFN1 on Cdc42 protein level in DM2 myoblasts; (C) Western blot detecting and quantitative analysis of the effect of overexpression of PFN1 on Cdc42 protein levels during differentiation; (D) the Cdc42 activity analysis in interfering or overexpression of PFN1 DM2 myoblast; (E) qRT-PCR detection of the mRNA expression level of differentiation marker genes in si-Cdc42 or si-NC transfected DM2 myoblasts; (F) qRT-PCR detection of the mRNA expression level of differentiation marker genes in pcDNA-Cdc42 or pcDNA3.1(+) transfected DM2 myoblasts; (G) Western blot analysis of differentiation marker protein levels in si-Cdc42 or si-NC transfected DM2 myoblasts; (H) Western blot detecting and quantitative analysis of differentiation marker protein levels in pcDNA-Cdc42 or pcDNA3.1(+) transfected DM2 myoblasts; (I) myotube formation status in si-Cdc42 or si-NC transfected DM2 myoblasts; (J) myotube formation status in pcDNA-Cdc42…

Figure 4. PFN1-Cdc42 negatively regulates bovine skeletal muscle via PAK/JNK signaling pathway: (A) Western blot testing and quantitative analysis of the effect of Cdc42 on PAK phosphorylation in si-Cdc42 or si-NC transfected DM2 myoblasts; (B) Western blot testing and quantitative analysis of the effect of Cdc42 on PAK phosphorylation in pcDNA-Cdc42 or pcDNA3.1(+) transfected DM2 myoblasts; (C) changes analysis of differentiation marker proteins of the DM2 bovine myoblasts with P-PAK inhibitor by Western blot; (D) protein level analysis of P-JNK after P-PAK inhibition in DM2 bovine myoblasts by Western blot; (E) Western blot detecting and quantitative analysis of the effect of PFN1 on the JNK signal pathway in si-PFN1 or si-NC transfected DM2 myoblasts; (F) Western blot detecting and quantitative analysis of the effect of PFN1 on the JNK signal pathway in pcDNA-PFN1 or pcDNA3.1(+) transfected DM2 myoblasts; (G) Western blot detecting and quantitative analysis of the effect of Cdc42 on the JNK signal pathway in si-Cdc42 or si-NC transfected DM2 myoblasts; (H) the effect analysis of Cdc42 on the JNK signal pathway in pcDNA-Cdc42 or pcDNA3.1(+) transfected DM2 myoblasts; (I) change analysis…