Tryple express 1

Cell counting kit-8 (CCK-8, Dojindo, Kumamoto, Japan) was used to investigate cell viability according to the manufacturer’ protocol. After digestion with cell dissociation solution TryPLE Express 1× (Invitrogen, Carlsbad, CA, USA), neurospheres were isolated into single cells, then resuspended with CM and cultured in PLL coated 96-well plates (2 × 10⁴ cells/well). After 24 h, the NSCs were pre-treated with the inhibitors for 2 h, and leptin (#400-21, Peprotech, Rocky Hill, NJ, USA) was then added to the medium to reach a final concentration. The working concentration of leptin were 25, 50, and 100 ng/mL, and/or the inhibitors were applied including the mitogen-activated protein kinase kinase-1 (MEK1) inhibitor PD98059 (#S1177, Selleck, Houston, TX, USA, 20 μM), phosphoinositide 3 kinase (PI3K) inhibitor LY294002 (#S1105, Selleck, Houston, TX, USA, 20 μM)
After another 48 h, about 10 μL cck-8 solution (water-soluble tetrazolium salt WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H tetrazolium]) was added into each well and then incubated at 37 °C. The optical density (OD) of formazan dye was detected by a microplate spectrophotometer (BioTek, Winooski, VT, USA) at 450 nm for 3 h of incubation. Results were averaged for 3 independent experiments, with each experiment containing at least 5 readings.

Human periosteum-derived mesenchymal stem cells (hPDCs) were isolated from periosteal biopsies of seven different donors, as described by Roberts et al.^{20 (link)}. Procedures were approved by the Ethical Committee for Human Medical Research (KU Leuven) and patient informed consent forms were obtained. The hPDC donors were expanded (5700 cells/cm²) at 37 °C, 5% CO₂ and 95% relative humidity in high-glucose GlutaMAX™ Dulbecco’s modified Eagle medium (DMEM; Life Technologies, UK) containing 1 × 10⁻³ m sodium pyruvate and supplemented with 10% irradiated fetal bovine serum (HyClone FBS; Thermo Scientific, USA) and 1% antibiotic–antimycotic (100 units/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B; Invitrogen). Medium was changed every 3–4 days. At all passages, cells were harvested with TrypLE™ Express 1× (Life Technologies, UK). In this study, all experiments and methods involving these cells were performed in accordance with the relevant guidelines and regulations.

The endothelial layer of corneal tissue from human cadaveric donors was peeled and digested in 2 mg/mL collagenase Type 1 (Thermo Fisher Scientific, Rochester, NY, USA) followed by centrifugation for 5 minutes at 1000 rpm. The pellet was re-suspended in TrypLE Express (1×) (Life Technologies, Monza, Italy) to dissociate single cells and centrifuged. The cells were re-suspended in 1 mL of the cell culture medium (1:1 Ham’s F12:M199 (Sigma-Aldrich, Gillingham, Dorset, UK)), 5% FBS, 20 µg/mL ascorbic acid (Sigma-Aldrich), 1% Insulin Transferrin Selenium (Gibco, Rochester, NY, USA), 10 ng/mL recombinant human FGF basic (Gibco), 10 µM ROCK inhibitor (Y-27632) (Miltenyi Biotech, Charlestown, MA, USA) and 1% PenStrep (Sigma-Aldrich) [36 (link)]. The HCEnCs were counted using a haemocytometer, and the number of plated cells was recorded. Lab-Tek II chamber slides were coated with 50 µL Fibronectin Collagen (FNC) coating mix (US Biological Life Sciences, Salem, MA, USA) for at least 15 minutes at 37 °C and 5% CO₂. The cells were plated, and the medium was replaced every alternate day until confluence.

In Co-IP experiments using HEK-293T cells, after exposed by blue light or kept in dark for 3 h, the transfected cells were washed with PBS pH7.4 (Gibco, 8118044), digested with TrypLE™ Express (1×) (Gibco,12605-028) at 37 °C for 5 min. The cells were then centrifuged at 800 g for 5 min and the supernatant discarded. The cell pellets were lysed using Pierce IP Lysis Buffer (87787, Pierce) with 1× EDTA-free Protease Inhibitor Cocktail Tablets (4693159001, Roche) and 1×PhosSTOP inhibitor cocktail (Roche) and incubated on ice for 15 min. The mixtures were then centrifuged at 14000 × g for 10 min at 4 °C to remove cell debris. The supernatant was mixed with 20 μL GFP trap beads or Myc agarose beads, incubated with vertical blending at 4 °C for 2 h. The beads-protein complex was washed 4 times with washing buffer [20 mM HEPES (pH 7.5), 40 mM KCl, 1 mM EDTA] and denatured by mixing thoroughly with 30 μL 4× Loading buffer and heating at 100 °C for 10 min^{16 (link)}.

Next, 36–48 h after transfection, the cells were exposed by blue light or kept in the dark for 3 h, washed twice with PBS buffer (cat # SH30256.01, HyClone, Logan, UT, USA), and then digested with TrypLE™ Express (1×) (cat # 12605-028, Gibco, Waltham, MA, USA) at 37 ℃ for 5 min. The cells were harvested for co-immunoprecipitation.
Cells transfected with different plasmid DNA were lysed by in 500 μL Pierce IP Lysis Buffer (cat # 87787, Pierce, Waltham, MA, USA) with 1× EDTA-free Protease Inhibitor Cocktail Tablets (cat # 4693159001, Roche, Waltham, MA, USA) and incubated on ice for 15 min. The mixtures were then centrifuged at 14,000× g for 10 min at 4 ℃ to remove cell debris. The supernatant was mixed with 20 μL GFP trap beads or Anti-DDDDk-tag mAb-Magnetic Agarose beads (cat # M185-10, MBL, Beijing, China), incubated with vertical blending at 4 ℃ for 2 h. The beads-protein complex was washed 4 times with washing buffer (20 mM HEPES (pH 7.5), 40 mM KCl, 1 mM EDTA) and denatured by mixing thoroughly with 30 μL 4× Loading buffer and heating at 100 ℃ for 10 min.

Vero (WHO) cells (ECACC no 88020401, equivalent to ATCC CCL81) (Nuvonis Technologies GmbH, Vienna, Austria), used for CelCradle^TM 500-AP experiments, were cultured in the serum-free medium OptiPRO SFM (Gibco, Grand Island, NY, USA) supplemented with 4 mM GlutaMAX (Gibco, Grand Island, NY, USA), 100 U/mL penicillin and 100 µg/mL streptomycin (Sigma, St. Louis, MO, USA) in T175 flasks (Nunc, Roskilde, Denmark) at 37 °C and 5% CO₂. Cells were detached with TrypLE Express (1×) (Gibco, Grand Island, NY, USA) for 5–10 min. The enzymatic reaction was stopped by dilution with cell culture medium, pelleting of cells by centrifugation for 5 min at 200× g, and subsequent resuspension in cell culture medium. The cells were sub-cultured every 3–5 days until passage 20.
Vero E6 cells (gift from J.D., University of Lille), used for TCID₅₀ assays, were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (high glucose, GlutaMAX Supplement, pyruvate) (Gibco, Paisley, Scotland) supplemented with 10% fetal bovine serum (Sigma), 100 U/mL penicillin and 100 µg/mL streptomycin (Sigma) in T175 flasks at 37 °C and 5% CO₂. Cells were detached with Trypsin-EDTA solution (0.05% w/v) (Sigma) for 5–10 min. The enzymatic reaction was stopped by dilution with serum-containing cell culture medium. The cells were sub-cultured every 2–4 days.