Il 10

Whole-cell lysates were prepared using 1x RIPA lysis buffer containing 50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholic acid, 1 mM EDTA (Millipore, Temecula, CA), and 1x protease inhibitor cocktail. Nuclear and cytoplasmic extracts were prepared using the NE-PER nuclear and cytoplasmic extraction kit according to the manufacturer’s protocol (Pierce, Rockford, IL). Protein extracts were resolved onto SDS-10% polyacrylamide gel (Bio-Rad, Hercules, CA) and transferred to nitrocellulose membranes (Thermo Fisher Scientific, Waltham, MA). The membranes were blocked with 5% milk in Tris-buffered saline/Tween-20 for 1 h at room temperature and then probed overnight at 4°C with an antibody specific to PU.1 (Cat# MA5-15064, Invitrogen, Carlsbad, CA), Ezh2 (Cat# 166609), IL-10 (Cat# sc-32815), S100A9 (Cat# sc-58706) (Santa Cruz Biotechnology), KDM6A (Cat# 33510S, Cell Signaling Technology, Danvers, MA) antibody. After washing, blots were incubated with the appropriate HRP-conjugated secondary antibody for 2 h at room temperature. Proteins were detected with the enhanced chemiluminescence detection system (Thermo Fisher Scientific), the bands were visualized using the ChemiDoc XRS System (Bio-Rad), and the images were captured with the Image Lab Software V3.0. The membranes were stripped and reprobed for β-actin (Invitrogen).

Recombinant WJSCs supernatants were collected 72 hours after transduction. Protein concentrations in the supernatants were determined using a BCA Protein Assay Kit (Thermo Fisher, USA). Then, protein (30 µg/lane) was loaded onto 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with protein marker and transferred onto nitrocellulose membranes (Bio-Rad, USA). The membranes were blocked using 5% non-fat milk and immunoblotting was performed using antibodies against IL-4 (Santa Cruz, USA), LIF (Abcam, UK), IL-10 (Santa Cruz, USA) and β-actin (Abcam, UK). Proteins of interest were detected using HRP-conjugated sheep anti-mouse IgG antibody (Abcam, ab6785). Finally, the protein band was visualized by chemiluminescence reagent (ECL) and the integrated optical density (IOD) of each protein band was measured. IOD values were adjusted against the internal standard, β-actin.

The following reagents were acquired from Sigma-Aldrich (St. Louis, MO, USA): AFB1, ionomycin, PMA, and RPMI-1640 medium. The fluorescently labeled antibodies targeting IFN-γ, STAT1, T-bet, IL-9, IRF4, IL-17A, IL-21, RORγT, STAT3, IL-22, AhR, TNF-α, IL-10, TGF-β1, FoxP3, and the buffers for red blood cell permeabilization/fixation were procured from BioLegend (San Diego, CA, USA). The Golgi-Plug and RORγT reagents were acquired from BD Biosciences (San Diego, CA, USA). The primary antibodies used in this study, including IFN-γ, T-bet, IL-9, IL-17A, RORγT, TNF-α, IL-10, and FoxP3, were acquired from Santa Cruz Biotechnology (Dallas, TX, USA). The FcR blocking reagent was acquired from Miltenyi Biotech (Bergisch Gladbach, Germany). The nitrocellulose membranes utilized in this study were acquired from Bio-Rad Laboratories (Hercules, CA, USA). The primers utilized in this work were acquired from GenScript (Piscataway, NJ, USA). The Merck, Darmstadt, Germany’s chemiluminescence kit was utilized to conduct Western blotting. The TRIzol^® reagent used in this study was obtained from Life Technologies (Carlsbad, CA, USA). The SYBR^® Green and cDNA kits utilized in this study were purchased from Applied Biosystems (Foster City, CA, USA).

Liver tissue levels of these factors were determined using sandwich Elisa protocol with antibodies or kits as follows: IL-1β (Cat # RAB0274, SigmaAldrich, USA), TNF-α (Cat # RAB0477, SigmaAldrich, USA), sirtuin 1 (Cat # sc-74465, Santa Cruz Biotechnology, Inc., USA), HO-1 (Cat # sc-390991, Santa Cruz Biotechnology, Inc., USA), IL-10 (Cat # sc-365858, Santa Cruz Biotechnology, Inc., USA), NF-kB (Cat # sc-8008, Santa Cruz Biotechnology, Inc., USA), Nrf2 (Cat # sc-722, Santa Cruz Biotechnology, Inc., USA), and TLR4 (Cat # SAB5700684, SigmaAldrich, USA).

Cells and tissue protein lysates were analysed by standard western blotting procedure, using antibodies such as PGE2, iNOS, TNF-α, IL-1β, IL-6, IL-10, NF-κB p65, ICAM-1, VCAM-1, P-selectin, and E-selectin obtained from Santa Cruz Biotechnology (Santa Cruz, CA) [27 (link)].

For tissue processing and histology, upon 30, 18, or 4 days after creating the wound, animals were euthanized, and the regenerated wound tissue was excised with a 10-mm biopsy punch (Acuderm, Fort Lauderdale, FL), fixed using 4% paraformaldehyde, and embedded by paraffin. The tissues were then sectioned and stained with H&E to measure granulation tissue thickness. The tissues were also stained with Masson’s trichrome to measure the epithelial thickness. The granulation tissue and epithelial thickness were quantified by measuring the thickness at five evenly spaced locations from the center of the wound for each animal using ImageJ. The average of the measurements obtained from each wound was calculated and compared among treatment groups.
Moreover, the tissue sections were stained for keratin-10, α-SMA, CD31, F4/80, IL-6, or IL-10 (Santa Cruz Biotechnology, Dallas, TX). The secondary antibodies were either conjugated to Alexa Fluor 488 or Alexa Fluor 555 (Invitrogen, Carlsbad, CA). Controls consisted of samples stained with the secondary antibody without incubation with a primary antibody. The development of neovascularization and changes in inflammation were quantified using ImageJ.