Fig. 2

Persistent FOS expression in muscle progenitor cells disrupts myogenic differentiation. A Experimental design. Fresh SCs were isolated from skeletal muscle (see “Materials and methods” section), infected with either pSLIK-Fos or pSLIK-Gfp viral vectors 1 day after isolation, selected with hygromycin (100 μg/ml) for 6 days, and then re-seeded at 4000 cells per 96-well in GM supplemented with 1 μg/ml of DOX for 48 h. Three hours before the end-point, cultures were pulsed with EdU. B Total number of Hoechst+ cells per well after 48 h in GM supplemented with 1 μg/ml of DOX (N = cells from 3 mice). C Percentage of EdU+ cells after 48 h in GM supplemented with 1 μg/ml of DOX (N = cells from 3 mice). D Representative histogram showing EdU(−) cells and EdU(+) cells, as defined by a fluorescent-minus-one control to set the negative and positive gates in flow cytometry. E Experimental design. Fresh SCs were isolated from skeletal muscle, infected with either pSLIK-Fos or pSLIK-Gfp viral vectors 1 day after isolation, selected with hygromycin (100 μg/ml) for 6 days, expanded in GM supplemented with DOX (1 μg/ml) for 48 h, and then 4000 cells were seeded per 96-well in DM (2.5% Horse Serum in DMEM) supplemented with DOX (1 μg/ml) and cultured for 72 h. F 20× image of pSLIK-Fos or pSLIK-Gfp myogenic cultures after 72 h in DM showing Hoechst+ (blue) and MyHC+ (magenta) cells. Scale bar represents 100 μm. G Quantification of the fusion index (total number of Hoechst+ nuclei in MyHC+ myotubes divided by the total number Hoechst+ nuclei) in pSLIK-Fos or pSLIK-Gfp myogenic cultures (N = cells from 3 mice). H Quantification using a differentiation index (sum of the integrated intensity of all Hoechst+ objects within MyHC+ objects divided by the sum of the integrated intensity of all Hoechst+ objects) in pSLIK-Fos or pSLIK-Gfp myogenic cultures (N = cells from 3 mice). B, C, G, H Mean comparisons using an unpaired, two-tailed, Student’s t test