Fig. 4

Impaired myogenic progression and differentiation and reduced DNA demethylation in Apobec2 knockdown myogenic precursors. a Time course of Apobec2 expression during differentiation of iPax7 ES cells in presence or absence of Pax7, which was induced by adding doxycycline (Dox) to the culture media. Apobec2 expression was measured by qRT-PCR (left), and by western blotting (WB) (right). Apobec2 mRNA expression was normalized by Gapdh expression (n = 3, mean ± SD) and as a reference the expression level in primary myoblasts (MB control) was measured (n = 2, mean ± SD). Myog and actin WB were used as controls for myogenic differentiation and equal loading, respectively. b DNA methylation and gene expression analyses of Myogenin were performed in a daily time course after Dox induction in the inducible myogenic model (n = 2). Each circle represents a CpG dinucleotide and its distance to the gene TSS is indicated below. The colour gradient represents the methylation level indicated in the legend. N.D. means non detectable. c Scheme of the experimental approach followed to knockdown Apobec2 in the inducible Pax7 model (left). Gene expression and protein level to evaluate Apobec2 knockdown efficiency using two independent clones were compared to control vector (right) (n = 3, mean ± SD; representative Apobec2 WB with the corresponding actin loading control). d Myogenin expression was measured in Pax7-induced myogenic precursors infected with Apobec2 shRNAs 4 days upon lentiviral transduction (right) (n = 3, mean ± SD). Representative images of Pax7-induced myogenic precursors infected with Apobec2 shRNAs under differentiation conditions stained with Myogenin and MHC antibodies. Scale bar: 100 μm. e DNA methylation analysis by sodium bisulphite sequencing of the Myogenin promoter in Pax7 ES-derived myogenic precursors transduced with Apobec2 shRNA1 and shRNA4 in three biological replicates. Each circle represents a CpG dinucleotide and its distance to the gene TSS is indicated below. The colour gradient represents the level of methylation indicated in the legend. Statistical significance with respect to the control samples is indicated with * for p-value < 0.05 applying the Kruskal-Wallis test. ESC embryonic stem cell, TSS transcription start site, shRNA short hairpin RNA