Calcipotriol

Alveolar epithelial cells type II (A549, ATCC) were maintained in DMEM medium supplemented with 10% FBS (Sigma-Aldrich, Burlington, MA, USA), 2 mM glutamine and 100 U/mL of penicillin and streptomycin (Lonza, Basel, Switzerland). We used the active form of vitamin D (1α,25-Dihydroxyvitamin D₃ or calcitriol) (cat.#D1530; Sigma-Aldrich; Vitamin D stock was 10 μM in ethanol) and the following vitamin D analogs (stocks were 50 μM in ethanol): calcipotriol (cat.#203537; Santa Cruz Biotechnology, Dallas, TX, USA), paricalcitol (cat.#477938; Santa Cruz Biotechnology), tacalcitol (cat.#sc-361371a; Santa Cruz Biotechnology, Dallas, TX, USA), 22-Oxacalcitriol (cat.#sc-361076; Santa Cruz Biotechnology, Dallas, TX, USA) and vitamin D2 (cat.#sc- sc-205988; Santa Cruz Biotechnology, Dallas, TX, USA). Treatments were performed in cells maintained in DMEM supplemented with 10% hormone-depleted serum. This serum was prepared by using the anion exchange resin AGR1-X8 from BIO-RAD (cat.#1401441; BIO-RAD, Hercules, CA, USA) as previously described [20 (link)]. Bleomycin sulfate (cat.# CAYM13877–50) was purchased to VWR [Radnor, PA, USA] (bleomycin stock: 50 mM in PBS).

Zymosan (Cayman chemical, Ann Arbor, MI, USA), Dexamethasone with ≥99% purity (Merck, Kenilworth, NJ, USA) and calcipotriol with >98% purity (Santa Cruz Biotechnology, Dallas, Texas, USA) were stored at -20° C until use. Dexamethasone and calcipotriol were prepared by mixing with 10 mg/ml lidocaine, (Orion, Espoo, Finland) and 99% ethanol containing the predetermined amount of the steroid hormone in a 96/4 volume ratio (lidocaine/ethanol) just before injections. The vehicle contained only lidocaine and ethanol in a 96/4 volume ratio without steroid hormones.