Zymography development buffer

HMC3 cells (800 k/well) were plated on six-well plates and, when confluence was reached, the monolayer was subjected to mechanical damage by multiple scratches using a sterile P200 pipette tip. After 24 h treatment, pellets were collected in RIPA lysis buffer (Sigma-Aldrich) supplemented with protease and phosphatase inhibitors. Fifty μg of each sample was separated in non-reducing conditions using Novex 10% Zymogram (Gelatin) Gels (Thermo Fisher Scientific), containing gelatin as a substrate. The gel was washed in renaturing buffer (2.5% TritonX-100) for 45 min at 25 °C and was then incubated with zymography development buffer (Bio-Rad Laboratories, Milan, Italy) for 45 min at 25 °C and maintained in fresh zymography development buffer for 22 h at 37 °C. The gel was then stained with 0.25% R-250 Coomassie solution (Sigma; 30 min at 25 °C) and destained in a 2:1 methanol:acetic acid solution (30 min at 25 °C). IBright 1500 (Thermo Fisher Scientific) was used to acquire digital scans of the gels. Images were subjected to densitometric analysis with the aid of the ImageJ processing software (https://imagej.nih.gov accessed on 2 August 2022).

Carnosine (L-carnosine), gelatin, and TTC (triphenyltetrazolium chloride) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Suture was purchased from Doccol Corp (Redlands, CA, USA). Gelatin-sepharose bead (Gelatin Sepharose 4B) was purchased from GE Healthcare (Chicago, IL, USA). Zymography renaturation buffer, zymography development buffer, and Coomassie Brilliant Blue R-250 was purchased from Bio-Rad (Hercules, CA, USA). Recombinant human MMP-2 was purchased from Merck Millipore (Burlington, MA, USA). Recombinant mouse MMP-9 was purchased from R&D Systems (Minneapolis, MC, USA). EnzChek Gelatinase/Collagenase Assay was purchased from Invitrogen (Carlsbad, CA, USA). Primary antibodies against ZO-1 and claudin-5, Alexa Fluor 555 donkey anti-rabbit, and Pierce BCA protein assay kit were purchased from Thermo Fisher Scientific (Rockford, IL, USA).