Fig. 7.

Quantitative analysis following abrogation of BMP signaling in cultured Pax7^CreERT2/+;RS6^+/− satellite cell-derived primary myoblasts. Experimental protocol: at day 0, satellite cells from Pax7^CreERT2/+ or Pax7^CreERT2/+;RS6^+/− adult mice were isolated using FACS and cultured in proliferation media; from days 2-4, cells remained either untreated (control), or were treated with 1 μM hydroxytamoxifen (4-OHT) or with 50 ng/ml of recombinant mouse Nog protein; at day 5, cells were either fixed for immunocytochemistry or collected for RNA extraction. (A,B) The dot plots (median indicated by the horizontal line) depict the relative mRNA copy numbers per 10³ Gapdh mRNA of human SMAD6 (A) or of the BMP target gene Id1 (B) from cultured satellite cells isolated from Pax7^CreERT2/+;RS6^+/− mice. (C) Cells were cultured at low density for a proliferation assay for the comparison of non-treated versus treated satellite cells isolated by FACS from skeletal muscles of Pax7^CreERT2/+ and Pax7^CreERT2/+;RS6^+/− mice. The number of cells per colony was counted from at least three wells per condition and per mouse (three mice); at least 50 colonies of cells per condition and per mouse were quantified. Data are shown as Whiskers-Tukey box plots. All P values were calculated using a t-test. (D-F) Following cultures of satellite cells from Pax7^CreERT2/+;RS6^+/− mice, the number of positive cells is given as a percentage of the total number of stained cells per colony following immunostaining against (D) myogenin (MyoG), (E) myosin heavy chain (MHC) and (F) Ki67. The quantification was performed on 13 to 20 colonies per culture (n=3 cultures, each derived from cells isolated from one mouse, total of n=3 mice). Data are shown as Whiskers-Tukey box plots. All P values were calculated using a t-test. In C-F, boxes indicate the interquartile range (IQR), the horizontal line indicates the median, whiskers indicate [1.5 × IQR] and dots indicate the outliers. (G,H) Dot plots (median indicated by the horizontal line) depict the relative mRNA copy numbers per 10³ Gapdh mRNA of (G) p21 and (H) p57 from FACS-isolated and cultured satellite cells from Pax7^CreERT2/+;RS6^+/− mice. Cells remained either untreated (control, n=4) or were treated with 1 μM 4-OHT (n=3) or 50 ng/ml of recombinant mouse Nog protein (n=3).