Halofuginone

The human keratocytes were cultured in serum-free medium as described above (with the negative control as the untreated human keratocytes). First, the upper concentrations of the reference bioactives were set according to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell-viability assay (see Supplementary Fig. 2). A reductionist corneal stromal wounding model was initiated by addition of 10 ng/ml TGF-β215 (link) (R&D systems, Minneapolis, USA) into the DMEM for 48 h (as the positive control). These ‘activated keratocytes’ were also treated with the different bioactives, which were added at the time of the ‘wounding’, with parallel incubation of the wound model for 48 h, due to the fast transient nature of these keratocytes. The bioactives used were: 10 ng/ml, 5 ng/ml recombinant IGF-1 (R&D systems, Minneapolis, USA), 10 ng/ml, 5 ng/ml halofuginone (Santa Cruz Biotechnology, Heidelberg, Germany), and 10 nM, 5 nM SAHA (Tocris Bioscience, Bristol, UK). The 10 ng/ml IGF-1 was also tested in combination with one concentration of each of the antifibrotics: 10 ng/ml halofuginone, and 10 nM SAHA. After completion of the first proliferative assays, the concentrations of halofuginone and SAHA used for the further experiments were chosen as 5 ng/ml and 10 nM, respectively. These human keratocytes were also used for further testing, as described below.

Cycloheximide (C1988, Sigma), 4′,6-diamidino-2-phenylindole (DAPI, D1306, Thermo Fisher), Halofuginone (sc-211579, SantaCruz), Nintedanib (S1010, Selleck Chemicals), Pirfenidone (P2116, Sigma), S3I-201 (SML0330, Sigma), Stattic (S7947, Sigma), U0126 (9903, CST), Thapsigargin (12758, CST), Tunicamycin (12819, CST).