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. 2018 May 18;9(1):292–304. doi: 10.1080/19491034.2018.1467722

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© 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — EDMD2 fibroblast medium induces TGF β2-dependent fibrogenic conversion in NHM cultures. (A) TGF β2 secretion in control and EDMD2 fibroblast culture medium. (B) Quantification of myofibroblasts in cultures of control human myoblasts maintained in medium conditioned by control (control) or EDMD2 (EDMD2) fibroblasts or treated with TGF β2 (control + TGF β2). The number of myofibroblasts was determined by counting alpha-SMA positive mononucleated cells (200 cells/sample were counted in three independent experiments). (C) Immunofluorescence staining of collagen I (green) and desmin (red) in cycling and differentiated NHM cultured in presence of medium conditioned by control or EDMD2 fibroblasts, treated (anti-TGFβ2) or not (NT) with anti-TGF β2 neutralizing antibodies. Nuclei were stained with DAPI. Bar: 10µm. (D) Immunofluorescence staining of ED-fibronectin (red) in cryosections of muscle tissue isolated from healthy donors (control), EDMD2 patients (EDMD2) or Becker muscular dystrophy patients (BMD). Nuclei were stained with DAPI. BMD tissue was used as positive control. In control muscle, ED-fibronectin is restricted to the area surrounding blood vessels. Bar: 10µm. (E) Quantitative analysis of proliferating cells in NHM cultures maintained in medium conditioned by control or EDMD2 fibroblasts, treated (anti-TGF β2) or not (NT) with anti-TGF β2 neutralizing antibodies. The number of proliferating cells was determined by flow cytometry. Means ± standard deviation are shown in graphs. Statistically significant differences are indicated by double asterisk (p<0.01) or triple asterisk (p< 0.001).