The Function of the Lysophospholipase 1 (LYPLA1) Gene in Fetal Sheep Myoblast Proliferation and Myogenic Differentiation

Figures

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  Figure 1. Expression analysis of MRFs in fetal sheep myoblasts at different differentiation time points (A–D): RT-PCR was used to detect the relative expression of MyoD1 (A), Myf5 (B), MyoG (C), and MRF4 (D) in fetal sheep myoblasts on days 1, 3, 5, and 7 of differentiation induction. Different lowercase letters indicate significant differences (p < 0.05).

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  Figure 2. Functional verification of the effect of LYPLA1 interference on fetal sheep myoblast proliferation. (A) RT-PCR detection of siRNA efficiency targeting LYPLA1 gene expression in fetal sheep myoblasts; (B) RT-PCR detection of relative expression levels of proliferation marker genes in fetal sheep myoblasts transfected with siRNA-364; (C) detection of cell viability using the CCK-8 assay in fetal sheep myoblasts following siRNA-364 transfection at 0 h, 24 h, 48 h, and 72 h; (D) EdU staining image of fetal sheep myoblasts transfected with siRNA-364; (E) effect of siRNA-364 transfection on the proliferation rate of fetal sheep myoblasts; (F,G) flow cytometry detection of the distribution of fetal sheep myoblasts at different cell cycle stages after transfection with siRNA-364; (H) percentage of fetal sheep myoblasts at different cell cycle stages after transfection with siRNA-364. * p < 0.05; ** p < 0.01; ns, not significant (p > 0.05); n = 3.

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  Figure 3. Functional validation of the effect of overexpression of LYPLA1 on the proliferation of fetal sheep myoblasts. (A) RT-PCR detection of the efficiency of pcDNA3.1(+)-LYPLA1 targeting LYPLA1 gene expression in fetal sheep myoblasts; (B) RT-PCR detection of the relative expression levels of proliferation marker genes in fetal sheep myoblasts transfected with pcDNA3.1(+)-LYPLA1; (C) detection of cell viability using the CCK-8 assay in fetal sheep myoblasts following pcDNA3.1(+)-LYPLA1 transfection at 0 h, 24 h, 48 h, and 72 h. (D) EdU staining image of fetal sheep myoblasts transfected with pcDNA3.1(+)-LYPLA1; (E) proliferation rate of fetal sheep myoblasts transfected with pcDNA3.1(+)-LYPLA1; (F,G) flow cytometry detection of the distribution of fetal sheep myoblasts at different cell cycle stages transfected with pcDNA3.1(+)-LYPLA1; (H) percentage of fetal sheep myoblasts at different cell cycle stages transfected with pcDNA3.1(+)-LYPLA1. * p < 0.05; ** p < 0.01; ns, not significant (p > 0.05); n = 3.

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  Figure 4. Functional verification of the effect of LYPLA1 gene interference on fetal sheep myoblast differentiation. (A–D) RT-PCR detection of the relative expression levels of MyoD1, Myf5, MyoG, and MRF4 on days 1, 3, 5, and 7 of induction differentiation of fetal sheep myoblasts transfected with siRNA-364; (E) inverted fluorescence microscopy observation of MyHC immunofluorescence staining on day 7 of induction differentiation of fetal sheep myoblasts transfected with siRNA-364. * p < 0.05; ** p < 0.01; ns, not significant (p > 0.05); n = 3.

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  Figure 5. Functional verification of the effect of LYPLA1 gene overexpression on fetal sheep myoblast differentiation. (A–D) RT-PCR detection of the relative expression levels of MyoD1, Myf5, MyoG, and MRF4 in fetal sheep myoblasts transfected with pcDNA3.1(+)-LYPLA1 on days 1, 3, 5, and 7 of induction differentiation; (E) inverted fluorescence microscopy observation of MyHC immunofluorescence staining in fetal sheep myoblasts transfected with pcDNA3.1(+)-LYPLA1 on day 7 of induction differentiation. * p < 0.05, ** p < 0.01, *** p < 0.001, ns p > 0.05, n = 3 biological replicates.