Fig. 4.

Inhibition of miR-27b activity in adult Pax3^GFP/+ satellite cells. (A) qRT-PCR amplification of Pax3 and MyoG transcripts from sorted Pax3^GFP/+ satellite cells after 3 (proliferating) and 5 (differentiating) days in culture. Results are normalized to GAPDH and relative to day 0. (B) qRT-PCR indicates mature miR-27b is up-regulated at 3 days, whereas miR-1 accumulates later at 5 days. (C) Protein levels of Pax3, Pax7, and Troponin T (TropT) were quantified by densitometry of Western blots after satellite cells were transfected with an anti miR-27b inhibitor (+) or control inhibitor (−) and cultured for 5 days. Pax3 levels increase in response to blocking miR-27b activity, confirming Pax3 as a direct target of the miRNA. Conversely, blocking the activity of miR-27b reduced the expression of Troponin T. Pax7, not predicted to be a target of miR-27, was unaffected. Results are indicated relative to β-tubulin expression and are from 2 independent Western blots. Representative immunoblots are shown. (D) Relative Pax3 transcript levels from cells transfected with control or anti-miR-27b inhibitors, normalized to GAPDH transcript levels.