Transforming growth factor β tgf β

Ex vivo culture of PCLS was performed at 37°C in 5% CO₂ for 96 h in the case of Poly(I:C) and/or IFNs and for 120 h in the case of fibrosis cocktail. IFN-γ (100 ng/ml, Fujifilm-Wako), IFN-α2 (100 ng/ml, R&D Systems), and Poly(I:C) (10 ng/ml, Tocris, Bristol, UK) were used to simulate RNA virus infections, such as SARS-CoV-2. The fibrosis cocktail consisted of 10 ng/ml platelet-derived growth factor (PDGF)-BB (Fujifilm-Wako), 10 ng/mL TNF-α (Fujifilm-Wako), 5 ng/mL transforming growth factor-β (TGF-β) (R&D systems), and 5 µM lysophosphatidic acid (LPA) (Focus Biomolecules, Plymouth Meeting, PA) and was replenished at 48 and 96 h (35 (link)). The O₂ concentrations for hypoxia and physioxia were used as 2 and 5%, respectively (39 (link), 40 (link)). PCLSs were incubated under hypoxia, physioxia, and normaxia (21% O₂) for 12, 24 and 48 h with or without Roxadustat (50 µM, Cayman, MI, USA) in hypoxia chamber (SV-140A, Blast, Tokyo).

Spleens were removed aseptically from the euthanized normal female BALB/c mice. The splenocytes were prepared with the lymphocyte separation medium as described above. Collected cells were washed twice with RPMI 1640 and resuspended in RPMI 1640 medium supplemented with 10% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich, MO, USA) for culturing experiments. Cell viability was evaluated by the trypan blue dye exclusion test.
Splenocytes (5 × 10⁶ cells/ml) in quintuplicate were seeded in 96-well plates supplemented with RPMI 1640 complete medium in a final volume of 200 μl. The cells were cultured with anti-CD3/anti-CD28 antibody (Abs) (both from eBioscience, San Diego, USA), IL-2 (PeproTech, London, USA), and transforming growth factor-β (TGF-β) (R&D Systems, MN, USA) in the presence or absence of IL-28B (5, 50, and 400 ng/ml) (R&D Systems, MN, USA) were added to the culture medium. Three days later, cells were harvested and analyzed for CD4, CD25, and Foxp3 expression by flow cytometry.

MUTZ-3 cells (ACC-295; DSMZ) were cultured in 12-well tissue culture plates (Corning) at a density of 0.5 × 10⁶ to 1.0 × 10⁶ cells/ml in minimal essential medium alpha (MEM-alpha) (Gibco) with 20% fetal bovine serum (FBS; HyClone, GE Healthcare), 1% GlutaMAX (Gibco), 10% conditioned supernatant from renal carcinoma cell line 5637 (ACC-35; DSMZ), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) at 37°C with 5% CO₂. We obtained MUTZ-3-derived Langerhans cells (muLCs) by differentiation of MUTZ-3 cells for 10 days in 100 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF; GenWay Biotech), 10 ng/ml transforming growth factor β (TGF-β; R&D Systems), and 2.5 ng/ml TNF-α (R&D Systems) as described previously (22 (link), 23 (link)). The phenotype of differentiated muLCs was verified by surface staining of CD34 (clone 581; BD Biosciences), CD1a (clone HI149; BD Biosciences), and CD207 (clone DCGM4, Beckman Coulter) using the respective antibodies and analysis by flow cytometry.
THP1 cells (TIB-202; ATCC) transduced with a lentiviral langerin construct or empty vector (EV) were cultured in RPMI (Lonza) supplemented with 5% FBS (Biowest), 1% GlutaMAX, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) at 37°C with 5% CO₂.

SFAC (containing saponin constituents higher than 74.4%), escin Ia (C₅₅H₈₆O₂₄, MW: 1131.26, purity ≥ 98%), escin Ib (C₅₅H₈₆O₂₄, MW: 1131.26, purity ≥ 98%), escin IIa (C₅₄H₈₄O₂₃, MW: 1101.23, purity ≥ 98%), escin IIb (C₅₄H₈₄O₂₃, MW: 1101.23, purity ≥ 98%), escin IIIa (C₅₅H₈₆O₂₃, MW: 1115.26, purity ≥ 98%) and escin IIIb (C₅₅H₈₆O₂₃, MW: 1115.26, purity ≥ 98%) were obtained from Yinxing Pharmaceutical Technology Co., Ltd. (Nanjing, China) (Figure 1); β-aminopropionitrile (BAPN) was obtained from Sa'en Co., Ltd. (Shanghai, China); Transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α) were obtained from R & D Systems (Minneapolis, USA); TRIzol and Lipofectamine 2000 reagents were obtained from Invitrogen (Carlsbad, USA); E-cadherin and LOXL2 antibodies were purchased from Genetex (Irvine, USA); HIF-1α and vimentin antibodies were purchased from BioWorld (Georgia, USA); α-SMA antibody was purchased from Abcam (Cambridge, UK); GAPDH monoclonal antibody was purchased from KangChen Bio-tech (Shanghai, China); HRP-conjugated secondary antibodies were purchased from Abbkine (Redlands, USA); Fetal bovine serum (FBS) was obtained from PAA (Linz, Germany). Other analytical reagent grade chemicals were obtained from Sinopharm Chemical Reagent Co. Ltd (Nanjing, China).

MCF-10A cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). MCF-10A cells were grown in Mammary Epithelial Cell Growth Medium (Lonza, Allendale, NJ, USA) supplemented with 5% horse serum. Recombinant epidermal growth factor (EGF) and transforming growth factor-β (TGFβ) were purchased from R&D Systems (Minneapolis, MN, USA).
The MAPK inhibitors CI-1040, SB203580, and SP600125 and AKT inhibitor LY29004 were purchased from Selleck Chemicals (Houston, TX, USA). BAY117082, dasatinib, doxorubicin, etoposide, paclitaxol and quercitin were purchased from LC labs (Woburn, MA, USA).

Cells from cell lines or freshly isolated cells (2 × 10⁵) were cultured in 6-well cell culture dishes (Corning, NY, USA) and RPMI-1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Recombinant exogenous S100A4 was added as indicated. If applicable, S100A4 was preincubated with a twofold molar excess of anti-S100A4 for 30 min at 4°C. S1P function was altered by using the S1P₁/S1P₃ antagonist VPC23019 (Cayman, Ann Arbor, MI, USA), S1P₂ antagonist JTE013 (Selleck, Shanghai, China), transforming growth factor-β (TGF-β) was purchased from R&D Systems (Minneapolis, MN, USA).