Bleomycin sulfate

Bleomycin sulfate was obtained from Hospira Inc. (Lake Forest, IL, USA), and neutralizing chicken anti-TGF-β1 antibody and control chicken IgG were obtained from R&D Systems (Minneapolis, MN, USA). Oleoyl lysophosphatidic acid (18:1 LPA) was obtained from Avanti Polar Lipids (Alabaster, AL, USA), and the cell lysis buffer was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Protease inhibitor cocktail tablets (EDTA-free Complete) were purchased from Roche Diagnostics (Indianapolis, IN, USA). Recombinant human TGF-β1 was obtained from Protech, Inc. (Rocky Hill, NJ, USA). Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG antibodies were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Rabbit anti-fibronectin and anti-β-catenin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Mouse anti-α-smooth muscle actin (α-SMA) and anti-β actin antibodies and Bay 11-7082 (NF-κB inhibitor) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The bleomycin‐induced skin fibrosis model was employed as described previously.29, 30 bleomycin was prepared by dissolving bleomycin sulfate (Hospira, Lake Forest, IL, USA) in sterile PBS. Briefly, 6‐month‐old WT and TRPV4 KO mice (n = 5 per group) received equal volumes (0.1 ml) of bleomycin (10 mg/kg) or PBS (control). All injections were administered subcutaneously to the shaved dorsal area of the mice every alternate day for 28 days. All mice were euthanized 24 hours after the last dose, and skin tissues were harvested for immunofluorescence staining. Skin samples were snap frozen in liquid nitrogen, embedded in OCT (Sakura Finetek, USA), and stored at −80°C. Cryostat sections (7 μm) were mounted on glass slides.