Fig. 5. CPEB1 regulates Myod1 protein output by acting on the CPEs within its 3’UTR.

a–c a Heatmap of the Log₂(Fold_Change) of muscle factors in the CPEB1 RIP-Seq analysis. b Real-time PCR (qRT-PCR) analysis of Myod1 mRNA after CPEB1 RNA-IP on fiSCs. (n = 2 independent experiments). c CPEB1 RIP-seq data of Myod1 transcripts. d CPEB1 immunostaining on QSCs, fiSCs and 4 hour cultured SCs (12 hours after mice were sacrificed). Nuclei were stained with DAPI. (n = 3 independent experiments). e Myod1 3′UTRs with wild-type (WT) or mutated cytoplasmic polyadenylation elements (CPEs) were inserted downstream of firefly luciferase (LUX) and co-transfected with pcDNA3.0-flag or pcDNA3.0-flag-CPEB1 into 293 T cells along with the Renilla luciferase vector as an internal control. Luciferase activity was quantified by dual-luciferase assay 36 hours after plasmid transfection. (n = 3 independent experiments). f The WT Myod1 3’UTR was inserted downstream of LUX and co-transfected with pcDNA3.0-flag or pcDNA3.0-flag-CPEB1 into 293 T cells along with the Renilla luciferase vector. One day after transfection, GFP or CPEB1 antibody (Ab) was transfected into 293 T cells. 4 hours after antibody transfection, the medium was replaced with fresh medium. One day afterwards, cells were harvested for dual-luciferase assay. (n = 4 independent experiments). g–j Myod1 protein expression analysis on SCs after CPEB1 antibody transfection. g Schematic illustration of Myod1 protein expression analysis on CPEB1 antibody transfected SCs. h GFP or CPEB1 antibody (Ab) was transfected into SCs. 4 hours after antibody transfection, SCs were subjected to Myod1 immunostaining. i Quantification of Myod1⁺ SCs (n = 3 independent experiments) and j Myod1 RFUs after antibody transfection (the number of quantified cells are 177 and 186 for the GFP Ab and CPEB1-Ab groups, respectively). Data are presented as mean ± SD in panels e, f, i, j. The p-values calculated by two-tailed unpaired t-test are used for comparing two groups in e, f, i, j ns not significant. Source data are provided as a Source Data file.