Tgf β1

The hepatic stellate cell line CFSC isolated from carbon tetrachloride-stimulated rats was provided by Professor Greenwell from the cell bank of George Washington University in the United States [8 ].The cells stored in liquid nitrogen were cultured in Dulbecco’s modified Eagle’s medium (DMEM; BI) supplemented with 6% fetal bovine serum (FBS; BI), 100 U/ml penicillin and 100 μg/ml streptomycin in humidified air at 37 °C with 5% CO₂ (Thermo). When the cells reached 80% confluence, they were dislodged by trypsinization and seeded in culture bottles.The cells were divided into the following groups: blank, TGF-β₁ (5 ng/ml), TGF-β₁ (5 ng/ml) + low,TGF-β₁ (5 ng/ml) + middle and TGF-β₁ (5 ng/ml) + high, with low, middle and high referring to the dose of calcium ionophore A23187. The blank group was cultured in DMEM without FBS for 48 h. The TGF-β₁ group was cultured with 5 ng/ml TGF-β₁ (Glico) for 48 h. The TGF-β₁ + low, middle, and high dose calcium ionophore A23187 groups were cultured with 5 ng/ml TGF-β₁ to which 1, 2 and 4 μM A23187 (Cayman) were added, respectively. The changes to each index were measured after 24 h.

Human GC cell lines (AGS and MKN-74) and Mouse Forestomach Carcinoma (MFC) cell line were purchased from the Procell Life Science and Technology (Wuhan, China). These cells were cultured in RPMI 1640 medium (Procell Life Science and Technology, Wuhan, China) with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA). Culture medium was changed every 2 days. Cells were pretreated with SR1078 (RORα activator, GC16392, GlpBio, Montclair, CA, USA), 3PO (3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one, PFKFB3 inhibitor, MCE, USA), DHEA (Dehydroepiandrosterone, G6PD inhibitor, MCE, USA) and glucose for 24 h. Cells were pretreated with TGF-β1 (Transforming growth factor beta 1, Cayman chemical, USA) for 48 h. SR1078 was dissolved in phosphate buffer saline (PBS). 3PO, DHEA, TGF-β1 and fluorouracil (GC14466, GlpBio, Montclair, CA, USA) were dissolved in dimethyl sulfoxide (DMSO).

Neurons were also treated with TGFβ1 (Lot. 0713354 D1014 from Acris GmbH, Germany) [physiological concentration: 2 ng/ml, (Fogel-Petrovic et al., 2007 (link))] or SB431542 (10 μM) (DaCosta Byfield et al., 2004 (link); Koo et al., 2015 (link)) (Lot. N° 0504746-44, Cayman Chemical Company, Michigan, United States); both TGFβ1 and SB431542 were added 18 h after plating.

Conditionally immortalized murine proximal tubular epithelial cells (TKPT) were used as previously described^{9 (link)} and cultured at 37°C in DMEM/F12 supplemented with 5% Fetal Bovine Serum (FBS) in a humidified incubator with 5% CO₂. TKPT have been authenticated and tested for mycoplasma contamination. Cells were seeded onto 60 mm Petri dishes. When reaching 50% confluency, cells were changed to serum-free DMEM/F12 for 12 h. Thereafter, cells were stimulated with different doses of TGF-β1 (0.5, 1, 2, or 4 ng/ml; R&D, Minneapolis, MN, USA) for 12 h, 24 h, or 48 h. Alternatively, cells were pretreated with different doses of TDZD-8 (2, 5, 10 μmol/L; Sigma-Aldrich, St. Loius, MO, USA), lithium chloride (LiCl, 2, 5, 10 mmol/L; Sigma-Aldrich) or forskolin (20 μmol/L, Cayman, Ann Arbor, MI, USA) for 30 min, and thereafter treated with TGF-β1 (2 ng/ml) for the indicated time. The eukaryotic expression vectors encoding the haemagglutinin (HA)-conjugated dominant-negative kinase-dead (KD), or constitutively active (S9A) mutant of GSK3β were transfected to TKPT as previously described^{9 (link)} by using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). After transfection, cells were subsequently subjected to the indicated treatment.