Tgf β1

Both Nestin-positive cells (Nes + cells) and Nestin-negative cells (Nes- cells) sorted from peritonea of Nestin-GFP mice were cultured. Transforming growth factor β1 (TGF-β1, MedChemExpress, NJ, United States) was used to stimulate the Nes + cells at 5 ng/mL for 48 h.

The human fetal lung fibroblast cell line (MRC-5) was purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies/Gibco, Grand Island, NY, USA) supplemented with 10 % (v/v) fetal bovine serum (Gibco) and 1% antibiotic-antimycotic solution (containing 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg amphotericin B; Sigma-Aldrich, St. Louis, MO, USA). Cells were maintained in a humidified air atmosphere with 5% CO₂ at 37 °C.
To induce the cell model of RA-ILD along with the animal model, recombinant TGF-β1 and IL-1β were added to the cell medium for 24 h, purchased from MedChemExpress (Monmouth Junction, NJ, USA). Chloroquine (CQ) and Bafilomycin A1 (BafA1) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). MG132 was purchased from Selleckchem (Houston, TX, USA).

The human type II alveolar epithelial cells (A549) were purchased from American Type Culture Collection (ATCC, Rockville, Maryland, USA). Cells grew in Dulbecco's modified Eagle's medium (DMEM, Hyclone, USA) supplemented with 10% fetal bovine serum (Gibco, MD, USA). Cells were incubated at 37°C with 5% CO₂. Cells were incubated with recombinant human TGF‐β1 (10 ng/mL, R&D Systems, Minneapolis, MN) for various time (0.25 hour, 0.5 hour, 1 hour, 2 hours, 24 hours or 48 hours). To induce the EMT cell model, A549 cells were maintained in growth media supplemented with 10 ng/mL TGF‐β1 for 48 hours, as previously described.22 ASV (Xiya Reagent, Shandong, China) was dissolved in DMSO at the final concentration of 100 μg/mL. Cells were also pre‐treated with TGF‐β1 inhibitor SB431542 (10 μmol/L, MedChemExpress, Monmouth Junction, NJ, USA) for 30 minutes or a specific PI3K/Akt inhibitor LY 294002 (20 μmol/L, MedChemExpress) before treatment with TGF‐β1 in some experiments.

NRK52E (ATCC, Manassas, VA, USA) cell line was cultured in a DMEM medium (Hyclone, Logan, UT). The cells were maintained at 37 °C under 5% CO2 in a humidified chamber. After incubation in the medium containing 1% FBS for 24 h, cells were treated with 10 ng/ml Recombinant Human TGF-β1 (Peprotech, NJ, USA) alone, 100 ng/ml relaxin alone or co-treated with TGF-β1 and relaxin for 48 h. To induce the Wnt/β-catenin pathway, NRK52E cells were treated with 40 μM SKL2001 (MedChemExpress LLC, Monmouth Junction, NJ, USA).

Human fetal lung fibroblast (HFL1) (Tongpai Biological Technology Co., LTD, Shanghai, China) were cultured in Ham's F12K medium containing 10% fetal bovine serum (FBS) (Sigma Aldrich, Saint Louis) at 37 °C with 5% CO₂. When cultured to 70–80% density, cells were randomly divided into four groups, including control group, TGF-β1 treated group, DHA treated group and TGF-β1+ DHA treated group. TGF-β1 treated group was treated with TGF-β1 (6 ng/mL, Sigma Aldrich), and the control group was treated with the same amount of solvent. DHA group were treated with DHA(30 μM, MedChemExpress) [26 (link)], and TGF-β1+DHA group were treated with TGF-β1 and DHA at the same time. The morphology of cells were observed under microscope IX51 (Olympus, Tokyo, Japan), and the cells were collected at 6h, 12h, 24h, 36h after treatments respectively and used for further detection.
The benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) [27 (link)], necrostatin-1 (Nec-1) [28 (link)] and Ferrostatin-1(Fer-1) [29 (link)] are specific inhibitor for apoptosis, necroptosis and ferroptosis respetively. To identify the form of death caused by DHA, DHA + Z-VAD-FMK(50 μM, MedChemExpress) [27 (link)] treated group, DHA + Nec-1(20 μM, Sigma-Aldrich, USA) [28 (link)] treated group, DHA + Fer-1(1 μM, Sigma Aldrich, USA) [29 (link)] treated group were replenished.