Recombinant human tgfb1

Primary human dermal fibroblasts were derived from forearm skin biopsies of 2 controls and 2 patients with SGS, as previously described in (21) . The fibroblasts were cultured in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) in the presence of antibiotics and passaged confluence. According to a previously described protocol with slight modifications (21), all cell culture experiments were conducted in serum-starved media for 24 hours prior to drug treatment. Stimulation was performed using 10 ng/ml recombinant human TGFb1 (R&D system). C646 dissolved in DMSO was treated at a dose of 20 µM for 24 hours of pretreatment before TGFb1 stimulation. SD208 dissolved in DMSO was treated at a dose of 10 µM for 24 hours of pretreatment before TGFb1 stimulation. Cells were collected at baseline, 6
hours after TGFb1 stimulation. The RNA was extracted from the cells using TRizol (ThermoFisher) via an RNeasy mini kit (QIAGEN), per the manufacturer's instructions.

FH535 (Tocris, 4344/10) was used at 15µM for in vitro experiments with DMEM + 2% serum as vehicle control. For in in vivo experiments, mice were dosed with 20mg/kg FH535 with PBS as vehicle control. Recombinant human BMP4 (100ng/ml; R&D, 314-BP), recombinant human TGFb1 (5ng/ml; R&D, 240-B), CHIR99021 (50nM-1uM; Stegment, 04-0004), LDN-193189 (250nM; Stegment, 04-0074-02), IWR1 (10uM; Tocris, 3532/10), IWP2 (5uM; Tocris, 3533/10) and TGFb Receptor I Inhibitor II (25nM; Tocris were also used in this study. A complete list of antibodies including source, clone name, product number and application are listed in Supplementary Table 1.