Fig. 1

Establishing a doxycycline-inducible system to ectopically express FOS in muscle progenitor cells. A Schematic of the doxycycline-inducible lentiviral system to express FOS or GFP. The all-in-one vector contains the gene of interest, the rtTA, and a selection marker in a single expression vector. The Ubiquitin-C (Ubi-C) promoter drives the constitutive expression of the rtTA, and hygromycin via an internal IRES sequence. In the presence of DOX (1 μg/ml), rtTA becomes activated and binds to the TRE promoter to drive expression of Fos or Gfp. B Experimental design schematic. 3000 SCs were seeded into wells containing GM, infected with virus expressing FOS or GFP, selected with hygromycin (100 μg/ml) for 6 days, rested for 2 days, and then 4000 cells were re-seeded into wells containing GM supplemented with 1 μg/ml of DOX. C Relative expression of Fos mRNA normalized to GAPDH in pSLIK-Fos muscle progenitor cells relative to pSLIK-Gfp muscle progenitor cells after 48 h in GM supplemented with 1 μg/ml of DOX (N = cells from 3 mice). D Relative expression of Fos mRNA (normalized probe signal intensity, microarray data [9]) in freshly isolated SCs relative to 5-day cultured SCs. E 20× images of cultured muscle progenitor cells (FOS+DOX and GFP+DOX) stained for FOS (Red) or Hoechst (nuclei), showing FOS-positive Hoechst-positive nuclei in cells infected with pSLIK-Fos virus. Scale bar represents 50 μm. F Corrected total cell fluorescence (CTCF) of FOS protein in individual pSLIK-Fos and pSLIK-Gfp muscle progenitor cells quantified in E (n = 1480 (FOS) and 1012 (GFP) cells. C Mean comparisons using an unpaired, two-tailed, Student’s t test and F using Mann-Whitney U test. Data represents mean ± SD