Pax3 expression is regulated by dicer and Pax3-3′UTR. (A) Pax3 expression was compared between wild-type myoblasts infected with a retrovirus vector expressing Pax3, Pax3-3′UTR, Pax3-3′UTR with mutations at miR-1/miR-206–binding sites (Pax3-3′UTRM), and a control empty vector by immunostaining (green). Nuclei were counterstained with DAPI (blue). (B) Pax3, Pax7, Bcl-2, and Bcl-xL gene expression were compared between wild-type myoblasts transfected with Pax3, Pax3-3′UTR, Pax3-3′UTRM, and a control empty vector by RT-PCR. (C) Relative Pax3 expression levels shown in B normalized by β-actin expression were measured. (D) After treatment with thapsigargin for 1 d, caspase-3 activity was compared between wild-type myoblasts infected with a retrovirus vector expressing Pax3, Pax3-3′UTR and Pax3-3′UTRM, and a control empty vector. (E) Myoblasts isolated from floxed dicer (dicer^fl/fl) mice were infected with adenovirus lacZ expression vector (control [Cont]) or adenovirus Cre/EGFP expression vector. GFP is only detected in myoblasts infected with adenovirus Cre/EGFP expression vector. (F) Control myoblasts infected with adenovirus lacZ expression vector shows the loxP band but not excised band after PCR. Infection with more adenovirus Cre/EGFP expression vector (10 and 100 µl) increased in amount of the excised band (Cre10 and Cre 100, respectively). (G) Cre, dicer, myogenic marker, and miRNA expression were compared between control dicer^fl/fl myoblasts infected with adenovirus lacZ expression vector and infected with adenovirus Cre/EGFP expression vector by RT-PCR. For miRNAs, all PCR products are ∼90-bp long. (H) ChIP assay with antibody against RNA polymerase II was performed. Input denotes each PCR product from naked myoblast genomic DNA. Pax3 and Pax7 but not Ig heavy chain (IgH) were detected by ChIP assay in wild-type (wt) and MyoD^−/− myoblasts. (B and G) β-Actin was monitored as a loading control. *, P < 0.05; **, P < 0.01. Error bars indicate SEM. Bars, 20 µm.