Figure 5.
Knockdown of m6A methylase genes regulates bovine myoblast differentiation. Bovine myoblasts were induced for myogenic differentiation 24 h after transfection with siNC or siRNAs (siFTO, siALKBH5, siMETTL3, siMETTL14 or siWTAP). (A) Fluorescence staining of MYHC protein (red) indicates myotube formation on day 3 of myoblast differentiation. Nuclei were stained with DAPI (blue). (B) The fusion index was calculated as the percentage of nuclei in fused myotubes out of the total number of nuclei. For each group, three random microscopic fields were selected randomly. (C) RT-qPCR was performed to detect the relative mRNA expression of MYOD1, MYOG, MYH3 and CKM on day 3 of myoblast differentiation. GAPDH was the normalization control. (D) Western blot assays demonstrating the expression levels of MYH3, MYOG, ACTN, FTO, ALKBH5, METTL3, METTL14, WTAP and GAPDH proteins in different treated cells. (B,C) Results are presented as the means ± SDs from three independent experiments. Using Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001 and ns = no significance.