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. 2013 Mar 18;5(4):626–639. doi: 10.1002/emmm.201202096

Figure 4.

Figure 4

Intrinsic differentiation potential and functional interactions between MuSCs and FAPs in response to HDACi in MDX mice at different stages of disease.

A. MuSCs isolated from young – 1.5 month old (upper panels) – and old – 1 year old (bottom panels) – MDX mice treated with ctrl or TSA (0.5 mg/kg) for 15 days, were isolated by FACS and cultured in vitro for 7 days in growth medium (GM) and then induced to differentiate in differentiation medium (5% HS) for further 3 days. Myogenic differentiation was assessed by MyHC immunofluorescnece. Representative images of MyHC (green) staining are shown. Nuclei were counterstained with DAPi (blue).

B. Graph showing the fusion index of the experiment represented in (A). We measured the percentage of nuclei that were MyHC− or MyHC+ in mononucleated myotubes (<2), the % of nuclei that were inside myotubes containing between 2 and 5 nuclei (2 << 5) and the % of nuclei inside myotubes containing more than 5 nuclei (>5). Data are represented as average ± SEM, n ≥ 2. Statistical significance tested by one-way ANOVA, *p < 0.05, **p < 0.01.

C. FAPs isolated from young – 1.5 month old (upper panels) – and old – 1 year old (bottom panels) – MDX mice treated with ctrl or TSA (0.5 mg/kg) for 15 days, were isolated by FACS and cultured in vitro for 7 days in growth medium (GM) and then induced to differentiate in adipogenic differentiation medium (adp-DM) for further 6 days. Adipogenic differentiation was assessed by Oil red staining. Representative images are shown.

D. Graph of the quantification of the number of Oil red O+ adipocytes per field in the same conditions showed in (C). Data are represented as average ± SEM of two different experiments (n ≥ 2); statistical significance assessed by t-test, *p < 0.05, **p < 0.01.

E. Scheme: MuSCs FACS-isolated from young MDX mice were co-cultured in transwell with FAPs isolated from either young MDX mice (ctrl or TSA treated for 15 days) or old MDX mice (ctrl or TSA treated for 15 days).

F. After 7 days of transwell co-culture the myogenic differentiation of satellite cells was assessed by immunostaining for MyHC (red). Nuclei were counterstained with DAPI (blue).

G. Graph showing the fusion index of the experiment represented in (F). We measured the percentage of nuclei that were MyHC− or MyHC+ in mononucleated myotubes (<2), the % of nuclei that were inside myotubes containing between 2 and 5 nuclei (2 << 5) and the % of nuclei inside myotubes containing more than 5 nuclei (>5). Data are represented as average ± SEM, n ≥ 2. Statistical significance tested by one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001.