Recombinant igf 1

Embryonic rat dorsal root ganglia (DRGs) were isolated as previously described by Greaves et al. (34 (link)). Whole-ganglion explants were plated onto poly-D-lysine- and Matrigel-coated wells (BD Biosciences) in 48-well plates and incubated in DRG medium (high-glucose DMEM supplemented with 0.01% penicillin-streptomycin, 10% fetal calf serum). Positive controls were supplemented with nerve growth factor (NGF) (Bio-Rad, Hercules, CA, USA) plus recombinant IGF-1 (R&D Systems, Minneapolis, MN, USA) (2–200 ng/ml) ± 125–500 nM Picropodophyllin (PPP; Tocris Bioscience, Bristol, United Kingdom). Some DRGs were incubated in PF or macrophage-conditioned medium diluted 1:1 in DRG medium ± 500 nM PPP. Images of explants were captured using an Axiovert microscope (Carl Zeiss, Oberkochen, Germany), an Axiovision camera, and software.

Recombinant CYR61 and SP600125 were purchased from Sigma-Aldrich (Lyon, France), recombinant IGF-1 was purchased from R&D Systems (Lille, France).
Neutralizing anti-IGF1 antibody was purchased from R&D Systems. Rabbit polyclonal anti-CYR61 and anti-pIGF1Rβ were purchased from Abcam (Cambridge, UK), anti-Actin was purchased from Sigma Aldrich and anti-IGF1Rβ, anti-pGSK3β, anti-pJNK, anti-JNK were purchased from Cell Signaling Technology (Saint Quentin en Yvelines, France). Mouse monoclonal anti-E-cadherin was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA), anti- GSK3β was purchased from Cell Signaling Technology, and anti-IGF1 was purchased from R&D Systems.

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.