Ly364947

C2C12-SKO cells were seeded in a 6-well plate and cultured in antibiotic-free DMEM medium containing 10% FBS overnight as described above. Next, the medium was changed to control medium and Stealth si-RNAs (Thermo Fisher Scientific) with a final concentration of 50 nM and prepared according to the manufacturer’s protocol. Then, each of four si-RNA platforms; 50 nM si-Control, 50 nM si-Tgf-β1, 50 nM si-Tgf-β3, or 50 nM dual si-Tgf-βs (50 nM si-Tgf-β1 + 50 nM si-Tgf-β3) was added to the medium of the cells (S1 Table). For transfection, we used 9 μl of Lipofectamine RNAiMAX (Thermo Fisher Scientific) per well (on day 1 and day 3). At the end of day 4, treated cells were used for immunohistochemistry or harvested for expression analyses.
For the assay that used the Tgf-β receptor inhibitor; LY-364947 (Fujifilm Wako, 123–05981), C2C12-SKO cells were seeded and cultured overnight in antibiotic-free DMEM with 10% FBS in a 6-well plate (day 0). On day 1, the medium was changed to differentiation medium containing 10 μM of LY-364947 or vehicle. The cells were treated twice with LY-364947 or vehicle every 48 hours in the differentiation medium (day 1 and day 3). At the end of day 4, treated cells were used for immunohistochemistry or harvested for expression analyses.

EdU (Sigma, 1 mg) was intraperitoneally injected into mice twice at 12 h intervals at 1 day before sacrifice. DMSO, PP2 (Millipore, 1 mM), NBI31772 (TOCRIS, 0.3 mM) and WAY 316606 hydrochloride (TOCRIS, 2 mM) in 100 μl were intradermally injected into mice twice a day for 4 days. IWP-2 (Sigma, 133.33 μM), Sfrp1 (R&D Systems, 20 μg/ml) and Igfbp2 (R&D Systems, 5 μg/ml) in 100 μl were intradermally injected into mice once a day for 4 days. LY364947 (Wako, 1.84 mM) in 100 μl was intradermally injected into mice once a day for 6 days.

AF488 N-hydroxysuccinimide (NHS) ester and AF647 NHS ester were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Rhodamine 6G (Rh6G) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Calcein AM was obtained from Dojindo (Osaka, Japan). SiR-Actin and the calcium channel inhibitor verapamil were obtained from Cytoskeleton (Denver, CO). The TGF-β receptor I inhibitor LY364947, PI, DX, and phenylmethylsulfonyl fluoride (PMSF) were obtained from Fujifilm (Tokyo, Japan). Rabbit polyclonal anti-fibronectin 1 antibody was purchased from Abcam (ab2413) (Cambridge, UK). pAcGFP1-Mem was purchased from Clontech (Takara Bio, Shiga, Japan). Smear Gell (product number: SG-01) for spheroid immunofluorescence was obtained from GenoStaff (Tokyo, Japan). The SlowFade mountant as a microscope was obtained from Thermo Fisher Scientific. The protease inhibitor cocktail (P8340) was purchased from Sigma-Aldrich.