Tgf β

Levels of interleukin (IL)‐1β, IL‐10 (PeproTech, Inc., Rocky Hill, Connecticut), and transforming growth factor (TGF)‐β (BD Biosciences, San Jose, California) were quantified by enzyme‐linked immunosorbent assay (ELISA) in lung tissue homogenate as instructed in the manufacturer's protocol, and normalized to the total protein content quantified by Bradford's reagent (Sigma‐Aldrich). Lung tissue was homogenized in lysis buffer (1X PBS, 0.01% Triton X, 1X Roche protease inhibitor cocktail [Roche Diagnostic, Mannheim, Germany]) using a bead mill (TissueLyser II, Quiagen, Hamburg, Germany) with a 3‐mm stainless steel bead (time: 5 minutes; frequency: 50 oscillations/s).

Human kidney tubular HK2 cells (ATCC, USA) were grown in DMEM/F12 containing 10% FBS (fetal bovine serum) and 1% penicillin/streptomycin (Gibco, USA). The culture was incubated at 37°C in 5% CO₂. The cells were treated with genistein (15 μM) for 24 h with or without TGF-β (BD Biosciences, USA, 5 ng/ml). Next, the cells were transfected with the synthesized human ALKBH5 siRNA oligonucleotides and the scrambled oligonucleotides at final concentrations of 50 nM using Lipofectamine 3,000 as per the methods described by the manufacturer. To generate ALKBH5 overexpression cells, HK2 cells were transfected with pcDNA/ALKBH5 or vector control through the liposome-mediated transfection. Transfected cells were subjected to drug treatments or not after 48 h of transfection. Finally, ALKBH5 expression was checked by conducting a western blot assay.

The spleens were extracted from Balb/c mice and naive CD4⁺ T cells were purified by using a magnetic cell sorting system (MACS® separation, Miltenyi Biotech, Bergisch Gladbach, Germany). The cells were cultured in RPMI 1640 culture media with 10% heat-inactivated FBS (Cellgro, Herndon, VA, USA), 100 U/mL penicillin (Cellgro), 0.1 mg/mL streptomycin (Cellgro), 2 mM L-glutamine (Cellgro), and 0.05 μM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, USA) at 37°C in a 5% CO₂-humidified incubator. Plate-bound anti-CD3 antibody (1 μg/mL, ebioscience) and anti-CD28 antibody (1 μg/mL, ebioscience) were used to stimulate T cells. Recombinant mouse IL-6 (25 ng/mL, BD, San Jose, CA, USA) and TGF-β (2.5 ng/mL, BD) were used for Th17 differentiation, and IL-2 (10 ng/mL, BD), IL-12 (5 ng/mL, Biosource, Camerillo, CA, USA), and anti-IL-4 (5 μg/mL) were added to induce differentiation into Th1 cells.

The antibodies used were mouse anti-E-cad IgG2a (BD Biosciences; San Jose, CA, USA), mouse anti-N-cad IgG1 and anti-TWIST1 (Santa Cruz Biotechnology; Santa Cruz, CA, USA), mouse anti-vimentin IgG1, rabbit anti-FN IgG and anti-GAPDH (Sigma-Aldrich), horseradish peroxidase (HRP)-labeled goat anti-mouse IgG, and HRP-labeled goat anti-rabbit IgG (Beyotime Institute of Biotechnology; Haimen, China). TGF-β was used, with 2 ng/mL as the final concentration (BD Bioscience). Other reagents were from Sigma-Aldrich unless described otherwise.

Lung and spleen viral titers at day 4 post-challenge were determined by counting plaques formed on the MDCK cells. The lungs and spleen were removed and homogenized in 1 ml of PBS containing antibiotics. The tissue homogenates were centrifuged, and the supernatants were tested for total viral load by standard plaque assay on MDCK cells and results were shown as PFU/ml. Simultaneously, inflammatory cytokines i.e. interferon gamma (IFN-γ), interleukin-6 (IL-6), and tumor growth factor beta (TGF-β) (BD Pharmingen, San Diego, CA, USA), and IgG/IgA levels (Southern Biotech, Birmingham, AL, USA) in lung supernatant were also analyzed by ELISA using specific paired antibodies48 (link).

Lung homogenates were centrifuged, and the supernatants were collected. Filtered cell-free lung homogenates were used to detect IL-1α, IGF-1, IL-13, CCL3, CCL5, CXCL1, CXCL2 (R&D Systems), IL-2, TGF-β, IL-4, IL-5, IL-6, IL-12p40, IFN-γ, CCL2 (B&D Biosciences), IL-10, IL-17, IFN-β, TNF and GM-CSF (Biolegend) by enzyme-linked immunosorbent assay (ELISA).