**5. Conclusions**

In conclusion, our data show that TGF-β inhibits myogenic gene expression in both myoblasts and myotubes, but does not affect myotube size in vitro. Most importantly, our results show that TGF-β stimulates *Col1a1* mRNA expression largely via autocrine expression of *Ctgf* and *Fgf-2*. Moreover, the effects of TGF-β on myogenic and fibrotic signalling are more pronounced than those of myostatin. Knockdown of *Tgfbr1* was sufficient to decrease *Ctgf* and *Col1a1* expression levels, while knockdown of *Acvr1b* had little effect. These results indicate that during muscle regeneration, TGF-β induces fibrosis via *Tgfbr1* by stimulating autocrine signalling of *Ctgf* and *Fgf-2.*

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/9/2/375/s1, Figure S1: TGF-β supplementation results in SMAD1/5 phosphorylation in C2C12 myoblasts and myotubes.

**Author Contributions:** Conceptualization, M.M.G.H., R.T.J. and W.M.H.H.; methodology, M.M.G.H. and W.M.H.H.; formal analysis, M.M.G.H.; investigation, M.M.G.H., R.A.G.C., C.O. and G.M.J.d.W.; resources, R.T.J. and W.M.H.H.; writing—original draft preparation, M.M.G.H.; writing—review and editing, M.M.G.H., R.A.G.C., R.T.J. and W.M.H.H.; visualization, M.M.G.H. and R.A.G.C.; supervision, R.T.J. and W.M.H.H.; project administration, W.M.H.H.; funding acquisition, W.M.H.H. and R.T.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Prinses Beatrix Spierfonds, grant number W.OR14-17.

**Acknowledgments:** We thank students M. Bulut and K. Doetjes for their contribution to this research.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
