Transforming growth factor β tgf β

MRC-5 human lung fibroblasts were obtained from American Type Culture Collection (Manassas, VA, United States) and seeded into 6-well plates at 1 × 10⁵ cells/mL in minimum essential medium (Hyclone, Logan, UT, United States) containing 10% fetal bovine serum (Gibco), 1% non-essential amino acids (Gibco), and 100 U/μg/mL penicillin/streptomycin (Gibco) at 37°C and 5% CO2 for 8 h. Next, Gal-9 (Biolegend, San Diego, CA, United States) or transforming growth factor-β (TGF-β) (Peprotech, Rocky Hill, NJ, United States) were added. The proliferation of MRC-5 fibroblasts stimulated with Gal-9 for 24 or 48 h was tested with a luminescent cell viability assay kit (Promega Corporation, Madison, WI, United States) according to the manufacturer’s instructions.

Isolated PBMCs were divided into six groups and cultured in different media at 1 × 10⁶ cells/ml: 10% control serum + 90% RPMI-1640 medium for the control, model, and hydrocortisone (HC) groups; 10% KJL containing serum + 90% RPMI-1640 medium for the KDH group; 5% KJL containing serum + 5% control serum + 90% RPMI-1640 medium for the KDM group; and 2.5% KJL containing serum + 7.5% control serum + 90% RPMI-1640 medium for the KDL group. Rat anti-CD3 mAb (BioLegend, Inc., USA) and rat anti-CD28 mAb (BD Pharmingen, USA) were used for activation of the T cell in the PBMCs. The 24-well culture plates for PBMC culture were precoated with 5 μg/ml anti-CD3 mAb at 4°C overnight. Anti-CD28 mAb was added into the medium at a final concentration of 2 μg/μl. Polarization towards Th17 cells was induced with 5 ng/ml transforming growth factor-β (TGF-β), 20 ng/ml IL-6, and 25 ng/ml IL-23 (all PeproTech, Inc., USA) in all groups except for the control group, to cause an imbalance between Th17 and Treg cells. For the HC group, 20 ng/ml HC was added into the medium. All groups of cells were cultured under conditions of 5% CO₂ and 37°C for 48 h prior to subsequent experiments.

Transforming growth factor-β (TGF-β; catalogue number: 100–21) was obtained from PeproTech (Rocky Hill, NJ, USA). We obtained harmine (catalogue number: HY-N0737A) from MedChemExpress (Monmouth Junction, NJ, USA). Anti-N-cadherin (14215S), anti-E-cadherin (3195P), anti-DYRK1A (2771), anti-p-Smad3 (9520), anti-Slug (9585) and anti-Snail (3879S) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-β-actin (47778), anti-Smad2/3 (133098), and anti-STAT3 (482) antibodies were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). The anti-p-STAT3 (Tyr705) (ab76315) and anti-tuberous sclerosis 1 (TSC1; ab270967) antibody was obtained from Abcam (Cambridge, Cambridgeshire, UK). The anti-α-tubulin (AF0001) antibody was purchased form Beyotime Institute of Biotechnology (Shanghai, China).

Cardiac differentiation of the isolated cells was induced following the protocol described by Smits and colleagues [12 (link)]. Briefly, cells were seeded with a density of 10⁵ cells per 6-well in hCSC medium. After 24 h, differentiation was induced with a cardiac differentiation medium consisting of a 1:1-mixture of IMDM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and Ham’s F12 nutrient mixture with GlutaMAX-I (Gibco), containing 10% horse serum (Dianova, Hamburg, Germany), 1x MEM nonessential amino acids (Bio Whittaker, Lonza, Basel, Switzerland) and 1x insulin-transferrin-selenium (Gibco). Then, 5 µM 5-azacytidine was added in three consecutive days and differentiation medium was refreshed at day 4. Six days after the start of the differentiation, ascorbic acid (Sigma Aldrich) was added every two days and 1 ng/mL transforming growth factor β (TGF-β) (Peprotech, Hamburg, Germany) was added twice weekly. Medium was refreshed every two to three days. After 28 days, the protein expression was analyzed by immunocytochemical staining for α-actinin as described above. As undifferentiated control, cells were cultured in hCSC medium.

TAE226 and PF‐562,271 were kindly provided by Novartis Pharm AG and Pfizer, respectively. The primary antibodies to the following proteins were purchased from Cell Signaling Technology and used for western blotting: FAK, IGF‐IRb, phospho‐IGF‐IRb (Tyr1135/1136), AKT, phospho‐AKT (Ser473), mTOR, phospho‐mTOR (Ser2448), S6 ribosomal protein, phosphor‐S6 ribosomal protein (Ser235/236), and cleaved poly‐ADP‐ribose polymerase (PARP). The phospho‐FAK (Tyr397) antibody was purchased from BD Pharmingen. An anti‐β‐actin antibody was used as a loading control. Recombinant human EGF, brain‐derived neurotrophic factor (BDNF), and transforming growth factor‐β (TGF‐β) were purchased from PeproTech. Recombinant human IGF‐I was purchased from Sigma‐Aldrich, and IGF‐IR inhibitor (picropodophyllin) was purchased from Santa Cruz Biotechnology.