Fig 1: MEF2C regulates miR222 transcription.a qPCR of MEF2C mRNA in the subcutaneous fat tissue of MSTN-KO and WT pigs (n = 3 biologically independent animals). b Western blotting of MEF2C protein in the subcutaneous fat tissue of MSTN-KO and WT pigs (n = 3 biologically independent animals). M is the Page Ruler Prestained Protein Ladder 26616 (ThermoFisher Scientific). c qPCR of miRNA222 abundance in response to the ectopic expression of MEF2C. n = 3 independent experiments. “***” Indicates statistically significant. d Western blotting of ectopic MEF2C expression. Flag, flag-tagged MEF2C. M is the Page Ruler Prestained Protein Ladder 26616 (ThermoFisher Scientific). e qPCR of miRNA222 abundance in response to MEF2C silencing. Data shown as the mean ± SD (n = 3 independent experiments). “***” Indicates statistically significant. f Western blotting of MEF2C silencing. M is the Page Ruler Prestained Protein Ladder 26616 (ThermoFisher Scientific). g Schematic of the MEF2C-binding sites on miR222 promoter region. “TATAAATACTT”, native binding motif. “GCCGCTGCACC”, introduced mutant binding motif. h Dual luciferase reporter testing of MEF2C upon miR222 promoter. pGL3-miR222-WT and pGL3-miR222-Mut represent wild-type and mutant version of miR222 promoter, respectively. n = 3 independent experiments. “***” Indicates statistically significant. i Dual luciferase reporter testing of MEF2C silencing upon miR222 transcription. n = 3 independent experiments. “***” Indicates statistically significant. j ChIP-PCR for the in vivo regulation of MEF2C on miR222 transcription. Sites A and B are the predicted MEF2C-binding sites in the miR222 promoter region. Anti-RNA, positive control antibody against RNA polymerase. Anti-MEF2C, antibody against MEF2C. IgG, negative control antibody. Input, sonicated DNA-protein mixture prior to immuno precipitation. PCR Neg. Ctrl and PCR Pos. Ctrl represent the negative and positive controls to demonstrate the primer specificity. M is 100 bp DNA ladder. k Sanger sequencing results for the PCR products from the lane “anti-MEF2C” of site A. l ChIP-qPCR validation of MEF2C binding at miR222 promoter. Anti-RNA and IgG are positive and negative controls, respectively. m MEF2C binding to the miR222 promoter verified in vitro by EMSA. The sequence and chemical modifications are shown at the bottom. WT probe (×150) was used as a competitor.