PDLIM5 Affects Chicken Skeletal Muscle Satellite Cell Proliferation and Differentiation via the p38-MAPK Pathway

Figures

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  Figure 1. The expression of PDZ and LIM domain 5 (PDLIM5) in different chicken tissues and chicken skeletal muscle satellite cells (SMSCs). (A) The expression of PDLIM5 in 11 different chicken tissues determined by qPCR (heart as the control group, n = 9). (B) The expression of PDLIM5 in chicken SMSCs at different time points determined by qPCR (n = 3). a,b,c,d p < 0.05.

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  Figure 2. PDLIM5 increased proliferation of chicken SMSCs. The mRNA expression of PDLIM5 in cells that transfected with si-RNA (A) or plasmid (B). After 48 h of PDLIM5 inhibition (C) or overexpression (D), qPCR was used to detect SMSC proliferation-related genes. Cell cycle analysis of SMSCs after inhibition (E) or overexpression (F) of PDLIM5 for 48 h. (G,H) After overexpressing PDLIM5 or inhibiting PDLIM5 in SMSCs, the cell proliferation activity was measured using CCK-8 kit. (I,J) The EdU fluorescence (red) was considered the proliferation indicator, and the cell nuclei were represented by DAPI (4’,6-diamidino-2-phenylindole, blue) fluorescence. The ratio of EdU-positive cells was determined when PDLIM5 was overexpressed or inhibited. * p < 0.05; ** p < 0.01 vs. NC and pcDNA3.1 (negative control). (n = 9).

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  Figure 3. PDLIM5 increased chicken SMSCs differentiation. (A,B) Gene expression of myosin heavy chain (MyHC), myogenic differentiation 1 (MyoD), and myogenin (MyoG) in SMSCs after overexpression or knockdown of PDLIM5. (C,D) Protein abundance of MyHC and MyoD in SMSCs after overexpression or knockdown of PDLIM5. (E) The immunofluorescence pictures and myotube area statistics of SMSCs after overexpression or knockdown of PDLIM5. In the picture, red represents myosin and blue represents stained nuclei. * p < 0.05; ** p < 0.01 vs. NC and pcDNA3.1 (negative control) (n = 9).

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  Figure 4. Interfering with transcriptome sequencing of si-PDLIM5 and negative control cells. (A) Volcano plot of statistically up-regulated or down-regulated genes after transcriptome analysis. (B) A heat map obtained by clustering analysis of differentially expressed genes that related to muscle development. (C) Analysis of differentially expressed genes by GO enrichment. (D) Analysis of the related pathways by KEGG enrichment. (E) Verification of mRNA expression of muscle development-related genes in transfected si-PDLIM5 or control. * p < 0.05; ** p < 0.01 relative to NC (negative control) (n = 9).

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  Figure 5. PDLIM5 regulated muscle development through the p38-MAPK pathway. (A) mRNA expression of MAPK pathway related genes transfected with si-PDLIM5 or pcDNA3.1-PDLIM5. (B) After overexpressing PDLIM5 in SMSCs and adding exogenous PDLIM5 and SB203580, the protein abundance of p38 and p-p38 was measured by Western blotting. In SMSCs with and without SB203580, the effect of PDLIM5 on cell proliferation (C) and differentiation (D) was determined. * p < 0.05; ** p < 0.01 vs. NC and pcDNA3.1 (negative control) (n = 9).

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  Figure 6. Schematic diagram of PDLIM5 regulating chicken skeletal muscle satellite cell proliferation and differentiation.