Figure 1. miR-206 is highly expressed in QSCs and regulates Pax3 transcript in SCs.

Quantitative RT-PCR analysis of Pax3 mRNA (A) or miR-206 (B) levels in QSCs sorted from uninjured muscle and from myoblasts (Mb) sorted from injured muscle 3.5 days after BaCl₂ injection. (C) Quantitative analysis of mRNA levels of Pax3 and Cyclophilin B (Pipb) in primary myoblast cultures treated with miR -1 (black) or miR-206 (white) in growth medium. (D) Quantitative analysis of Pax3 mRNA in primary myoblast cultures treated with anti-miR-1 (black) or anti-miR-206 (white), then cultured in differentiation medium for 1 or 2 days.(E) Luciferase reporter assays showing the long form of Pax3 3′UTR repression by miR-206 in 293 cells. Luciferase constructs and miR-206-expressing plasmid were co-transfected in 293 cells, and luciferase activity was measured 48 hours post-transfection. Mutation of both target sites is necessary to abolish the repression of luciferase activity by miR-206 (m1+m2). (F) Luciferase reporter assays showing the long form of Pax3 3′UTR repression by miR-206 in C2C12 cells after differentiation. After transfection with luciferase constructs, C2C12 cells were cultured in differentiation medium for 48 hours to allow endogenous miR-206 upregulation. Mutation of both target sites is necessary to abolish the repression of luciferase activity by miR-206 (m1+m2). Pax3 murine long 3′UTR was appended to the luciferase ORF (Luc). The different luciferase constructs are indicated on the left of the graphs (WT, m1, m2, m1+m2). miR-206 complementary sites (206₁and 206₂) (vertical line) and mutated sites (cross) are indicated.(G) Competitive inhibition of miR-206 using the Pax3 3′UTR construct. Immunoblot analysis of Pax3 protein level in satellite cell-derived myoblasts, 48 hours after transfection with luciferase constructs containing either wild-type or mutated miR-206 target sites, then cultured in differentiation medium. Repression of Pax3 transcript by miR-206 was rescued by overexpressing wild-type Pax3 3′UTR construct, which acts as a competitive inhibitor (* p<0.05; ** p<0.001; N.S. - not significant; n=3). See also figure S1.