Fig 1: Spatiotemporal expression patterns of the CFL1 gene in Qinchuan cattle. Relative spatial expression level was analyzed by qRT-PCR in different tissues of three growth and development stages: Qinchuan fetal bovine (A), Qinchuan calf (B), Qinchuan adult cattle (C). The expression levels of lung tissues were considered as 1. Temporal expression patterns of the CFL1 gene in different tissues of Qinchuan cattle. Relative temporal expression levels in three periods and seven tissues of Qinchuan cattle were investigated using qRT-PCR (D,E). The expression levels of all tissues in fetal bovine were considered as 1. The mRNA expression level of CFL1 was normalized to GAPDH. Data are means ± SE of n = 3 independent experiments, each performed in triplicate. *, p < 0.05 and **, p < 0.01, two-tailed t test.
Fig 2: Construction of adenovirus overexpression and interference vectors of the CFL1 gene. Expression efficiency of CFL1 was detected by qRT-PCR (A,B) and western blot (C) in HEK293 cells after being transfected with adenovirus overexpression (CFL1-CMV) and interference (shCFL1-1 and shCFL1-2) vectors for 48 h. The C2C12 cells were induced to differentiate for 4 days after being transfected with CFL1-CMV or shCFL1-2, and the relative expression of myoblast differentiation marker genes MYOD, MYOG, and MYH3 were detected by qRT-PCR (D,E). Expression trends of the CFL1 gene and myogenesis factors were detected in C2C12 cells after differentiation had been induced for 1, 2, 4, 6 and 8 days by 2% horse serum (F). The mRNA expression level was normalized to GAPDH. Data are means ± SE of n = 3 independent experiments, each performed in triplicate. *, p < 0.05 and **, p < 0.01, two-tailed t test. CTRL, Control. GM, growth medium. DM, differentiation medium.
Fig 3: Effects of the CFL1 gene on the differentiation of bovine primary myoblasts. Expression of CFL1, MYOD, MYOG and MYH3 were detected by qRT-PCR after being transfected with CFL1-CMV into bovine primary myoblasts and induced for 1, 3, 5, 7 days by 2% horse serum (A–D). Expression of CFL1, MYOD, MYOG and MYH3 was detected by qRT-PCR after being transfected with shCFL1-2 into bovine primary myoblasts and induced for 1, 3, 5, 7 days by 2% horse serum (E–H). Expression trends of CFL1 gene and myogenesis factors were detected by qRT-PCR after being transfected with CFL1-CMV or shCFL1-2 into bovine primary myoblasts and induced for 1, 3, 5, 7 days by 2% horse serum, the expression levels of four genes in GM were considered as 1 (I,J). The mRNA expression level was normalized to GAPDH. Data are means ± SE of n = 3 independent experiments, each performed in triplicate. *, p < 0.05 and **, p < 0.01, two-tailed t test. CTRL, Control. GM, growth medium.
Fig 4: Regulatory relationship between miR-182 and the CFL1 gene. Expression efficiency of miR-182 was detected by qRT-PCR in C2C12 cells after being transfected with miR-182 mimic and inhibitor for 48 h (A). CFL1 mRNA (B) and protein (C) expression in bovine primary myoblasts were detected by qRT-PCR and western blot after being transfected with miR-182 mimic, miR-182 inhibitor and NC for 48 h. Sequence of miR-182 and its predicted binding site in CFL1 3'UTR and mutation 3'UTR (D). The miR-182 mimic or NC co-transfected with CFL1-Luc and CFL1-del Luc into bovine primary myoblasts individually, and Renilla luciferase activity was normalized to the firefly luciferase activity (E). The expression of myoblast differentiation marker genes MYOD, MYOG, and MYH3 were detected by qRT-PCR after being transfected with miR-182 mimic or inhibitor into C2C12 cells and induced for 4-day by 2% horse serum (F,G). The expression level of miR-182 was normalized to U6, the mRNA expression levels of CFL1, MYOD, MYOG, and MYH3 were normalized to GAPDH. Data are means ± SE of n = 3 independent experiments, each performed in triplicate. *, p < 0.05 and **, p < 0.01, two-tailed t test. NC, Negative Control; means.
Fig 5: Proposed model of how the CFL1 gene regulates the differentiation of bovine primary myoblasts.
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