Netrin 1

Netrin-1 was purchased from R&D Systems (1109-N1-025, Minneapolis, MN) and BSA from MultiCell (500-0206). They were used at 100 ng/ml. Unless specified, Netrin-1 stimulations were performed for 5 min. EHT1864 was purchased from Selleckchem (S7482, Batch: 5748201). TRITC-phalloidin was purchased from Sigma-Aldrich (P1951), and Alexa Fluor 488-phalloidin was purchased from Thermo Fisher Scientific (A12379).

The assay was performed as in Schmidt, Clavarino, Ceppi, and Pierre, 2009^{32 (link)}. Cultures were incubated in 10 µg/mL puromycin (ThermoFisher) for 10 min increments concurrent with or following treatment with Slit-2 (200 ng/mL), netrin-1 (100 ng/mL), or ephrin-A1 (200 ng–2 µg/mL) (R&D), and pharmacological agonists/antagonists as noted in the results, including rapamycin (Calbiochem) and anisomycin (MP Biomedicals). puromycin-containing media was rinsed 2× with NB media before fixation. Growth cones were fixed and stained with anti-puromycin primary antibody (1:1000, PMY-2A4 was deposited to the DSHB by Yewdell, Jonathan (DSHB Hybridoma Product PMY-2A4)) and AF488 secondary (ThermoFisher) with AF-conjugated phalloidin (ThermoFisher) counterstain and analyzed as described above. All growth cones were normalized to TSC2^+/+ puromycin-only control.

Mice primary VSMC seeded on micropillar array were washed twice using normal physiological saline solution (NPSS) with 140 mM sodium chloride, 5 mM potassium chloride, 2 mM magnesium chloride, 1 mM calcium chloride, 10 mM HEPES, and 10 mM glucose. Cells were then loaded for 20 mins with calcium probe Fluo-4 (F14201, Thermo Fisher Scientific) followed by two washes with NPSS. Imaging was performed using a Zeiss Axio observer with a ×20 objective excited at 480 nm. Before recording data, Ca²⁺ signal was stabilized by imaging VSMC continuously every 1 s for 6 min. To image Ca²⁺ in VSMC upon mechanical stimulation, VSMC were imaged every 200 ms and mechanical stimulation was applied 3 s from the start of the recording. To image Ca²⁺ in VSMC with drug stimulation, Yoda1 (#21904, Cayman Chemical) or recombinant mouse Netrin-1 (#1109-N1, R&D Systems) at a final concentration of 30 µM and 2.5 µg/ml were respectively added to VSMC media. For Piezo1 inhibition assays, cells were preincubated with 2.5 µM GsMTx4 (#STG-100, Alomone labs) for 10 min or 10 µM Dooku1 (#6568, R&D Systems) for 30 min prior to stimulation.

Dorsal spinal cord explants from E12.5 embryos were dissected and cultured in collagen gels as described previously (Serafini 1994). Briefly, explants were cultured in 50% OptiMEM (Gibco, #31985-070) and 45% Ham’s F-12 (Gibco, #11765-054) media supplemented with 5% horse serum (HS, Gibco, #16050122), 0.75% glucose (Thermo, #D16-500), and 1X penicillin/streptomycin/glutamine for 48 hr with 500 ng/ml Netrin-1 (R&D, #1109-N1/CF).

We performed the axon cultures as described above for RNA-seq analysis. Then, we treated the eyes with lysine- and arginine-free L15 (60%) medium for 1hr. After eye removal to eliminate the cell bodies, the axons were cultured in L15 depletion medium containing “heavy” amino acids (84 μg/ml [13C6,15N4] l-arginine, 146 μg/ml [13C6,15N2] l-lysine (Silantes, Germany) and Netrin-1 (600 ng/ml; R&D systems) for 3hrs. Soma removal was confirmed by absence of nuclear DAPI staining. For the preparation of control eye samples, we cultured dissected eyes in L15 depletion medium containing “heavy” amino acids at room temperature for 48hrs. Lysis of axons was performed using 500 μL Lysis buffer (9mM Tris-HCl pH 7.4, 270mM KCl, 9 mM MgCl₂, 1% n-octylglycoside (Sigma-Aldrich),100μg/ml cycloheximide (Sigma-Aldrich), 0.5mM DTT, EDTA-free protease inhibitor cocktail (Roche) and SUPERase In RNase Inhibitor (Ambion)). Lysates were centrifuged at 16.000 g at 4°C for 15 min and the supernatant was transferred to an ice-cold 1.5ml tube. For the puromycin/RNaseA/T1 treated sample, axons were treated with 200 μM puromycin for 15min before lysis and lysates were treated with 10 μg/μl RNase A (Ambion) and 250U RNase T1 (Ambion) for 15 min at 25°C.

Mouse entire corneal epithelium, including the limbal region, was marked with 3 mm trephine and subsequently scraped with a corneal rust ring remover (Alger Co, Lago Vista, TX) in anesthetized normal and diabetic mice. After the corneal epithelium scraped, diabetic mice were injected subconjunctivally with 50 ng netrin-1 (5 μL/eye, R&D Systems, Minneapolis, MN) or equal phosphate buffer saline (PBS) as the vehicle control. For topical netrin-1 application, netrin-1 (50 ng in 2.5 μL PBS) was dropped onto the corneal surface by a 10 μL tip four times daily per eye for 3 days. For netrin-1 receptor inhibition, A2BAR antagonist PSB1115 (2.5 μg in 5 μL PBS, R&D Systems) or UNC5B receptor-blocking antibody (5 μg in 5 μL PBS, R&D Systems) was injected subconjunctivally at 24 h before and 0, 24, and 48 h after the scrape of corneal epithelium. Ofloxacin eye drops were applied to all mice to avoid infection. After 24, 48, and 72 h, corneal epithelium defects were visualized by staining with fluorescein sodium and photographed under slit lamp microscope (BQ900; Haag-Streit, Bern, Switzerland). The stained area on the residual epithelial defect was analyzed by using Image J software (National Institutes of Health, Bethesda, MD) with the original epithelial defect area as 100%.