Figure 3: Satellite cells in miR-431 TG mice have Pax7^Lo characteristics.

miR-431 TG mice were generated in which transgene expression was driven by the β-actin promoter (pCAGGS). Two lines of the TG mice (H and O) were used. Values are mean±s.e.m. or one representative value from four to five pairs of WT and TG mice. *P<0.05, **P<0.01. NS, not significant. Two-tailed Student’s t-test was used for all statistical testings. (a,b) miR-431 overexpression (OE) levels in the skeletal muscle of the H (a) and O (b) lines were examined using real-time RT–PCR with TaqMan probe. U6 snRNA was the internal control. Pax7 expression in the same samples was analysed using real-time RT–PCR and GAPDH was the internal control. (c) Number of satellite cells in cryosections of TA muscle, stained for Pax7, from miR-431 TG mice and WT controls. (d) Satellite cells were genetically labelled with GFP by crossing miR-431 TG mice with Pax7Cre::RosamTmG reporter mice. GFP⁺ cells from the skeletal muscle of Pax7-Cre::RosamTmG::miR-431 mice and Pax7-Cre::RosamTmG controls were sorted using FACS. (e) miR-431 OE in FACS-sorted satellite cells from d was determined using real-time RT–PCR. U6 snRNA was the internal control. Pax7 expression in the same samples was determined using real-time RT–PCR and GAPDH was the internal control. (f) Satellite cells sorted from the skeletal muscle of Pax7-Cre::RosamTmG mice were stained with Pax7, and the stained cells were sorted with FACS into Pax7^Hi and Pax7^Lo subpopulations. (g) Expression levels of miR-431 and Pax7 in the Pax7^Hi and Pax7^Lo satellite cells from f were analysed using real-time PCR. (h) Satellite cells were sorted by FACS to select CD31-CD45-Sca1-VCAM+ from miR-431 TG and WT mice and stained with Pax7; the stained satellite cells were further sorted by FACS to calculate Pax7^Lo subpopulations. (i) The percentage of the Pax7^Lo subpopulation were calculated based on h,j. Overall profile of Pax7⁺ cells sorted in h,k. Expressions of the stemness-, differentiation- and mitochondria-related markers, determined using real-time RT–PCR in sorted satellite cells from miR-431 TG and WT mice.