Mitomycin c

For RNA-seq and immunofluorescence studies, corneas received one topical application (20 μl per eye) of 0.02% Mitomycin-C (#3258, Tocris) dissolved in PBS at the time of wounding (MW) or 18 hr prior to sacrifice in the control unwounded mice (MC). After 2 minutes, excess solution was removed with a Kimwipe and by blinking. For trephine wounded corneas, the same volume (20 μl/eye) and concentration of MMC was applied at the time of and 18 hr after wounding.

Sterile 2-well culture inserts (Ibidi GmbH, Gräfelfing, Germany) were placed into 12 well culture plates. Cells were seeded at 50,000 cells into each side of the Ibidi insert then treated with designated siRNA for 24 h. Ibidi inserts were removed to create ‘cell-free gap’. Mitomycin C (1 µg/ml; Tocris, Abingdon, UK) was added to designated treatment conditions to prevent cell proliferation from confounding the cell migration results. Regions of interest (cell-free gap area) were immediately defined after Ibidi insert removal through CellSens software (Olympus, Essex, UK). Defined regions of interest were imaged at 0, 24, and 48 h. Images were collected on an Olympus IX83 inverted microscope using a 4x objective and captured using an Orca ER camera (Hamamatsu, Hertfordshire, UK). Images were then processed, and cell-free area was quantified using Fiji ImageJ (http://imagej.net/Fiji/Downloads; Method S4). % Gap closure was calculated as follow: % gap closure = ((gap area at 0 h−gap area at × h) ÷ gap area at 0 h) × 100

Human embryonic stem cells (hESCs) were differentiated with a modified dual-SMAD inhibition protocol towards floor plate-based midbrain dopaminergic (mDA) neurons as described previously^{64 (link)}. Briefly, hESCs were maintained on mouse embryonic fibroblasts and passaged with Dispase (STEMCELL Technologies). For each differentiation, hESCs were harvested with Accutase (Innovative Cell Technology). At day 30 of differentiation, hESC-derived mDA neurons were replated and maintained on dishes precoated with polyornithine (PO; 15 μg mL⁻¹), Laminin (1 μg mL⁻¹), and Fibronectin (2 μg mL⁻¹) in Neurobasal/B27/L-glutamine-containing medium (NB/B27; Life Technologies) supplemented with 10 μM Y-27632 (until day 32) and with brain-derived neurotrophic factor (BDNF; 20 ng mL⁻¹; R&D), ascorbic acid (AA; 0.2 mM, Sigma), glial cell line-derived neurotrophic factor (GDNF; 20 ng mL⁻¹; R&D), transforming growth factor type β3 (TGFβ3; 1 ng mL⁻¹; R&D), dibutyryl cAMP (0.5 mM; Sigma), and DAPT (10 nM; Tocris). Two days after replating, mDA neurons were treated with 1 μg mL⁻¹ mitomycin C (Tocris) for 1 h to kill any remaining non-post mitotic contaminants. Toxicity assays were performed at 65 days of neuron differentiation. PU-AD was added to the cells for 72 h and the CellTiter-Glo Luminescent Cell Viability Assay (Promega) assay was performed according to the manufacturer’s indications.