U0126

U251MG, A549, MCF-7 and 293T cells (The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences) were used in the present study. All cell lines were cultured at 37°C with 5% CO₂. Ham's F-12K (Kaighn's) Medium (Thermo Fisher Scientific, Inc.) was used to culture A549 cells. U251MG, 293T and MCF-7 cells were cultured in DMEM with high glucose (HyClone; Cytiva). All media were supplemented with 10% FBS (Shanghai ExCell Biology, Inc.) and 100 µg/ml penicillin and streptomycin to make complete medium. Media were changed every other day. The inhibitors and activators used in the present study included an ERK inhibitor (FR180204; 10 µM, 24 h; cat. no. S7524; Selleck Chemicals), a 90 KDa ribosomal protein S6 kinase (RSK) inhibitor (SL0101; 50 µM, 24 h; cat. no. 77307-50-7; Sigma-Aldrich; Merck KGaA), vemurafenib (2 µM, 24 h; cat. no. A3004; APeXBIO Technology LLC), a MEK inhibitor U0126 (2 µM, 24 h; cat. no. 9903; Cell Signaling Technology, Inc.), TGF-β (10 ng/ml, 48 h; cat. no. 8915; Cell Signaling Technology, Inc.) and 20 mM Citrate pH 3.0 (Sterile) (cat. no. 9871; Cell Signaling Technology, Inc.). To examine TGF-β inducing EMT, U251 cells were incubated with 10 ng/ml TGF-β, and the control group was incubated with an equal volume of 20 mM citrate solution buffer, which was the buffer solution for TGF-β.

The hPDLSCs were plated into 48-well plates (4×10
⁴ cells/well), 24-well plates (6×10
⁴ cells/well) and 6-well plates (2.5×10
⁵ cells/well) to perform ALP staining, ARS staining and western blot/qPCR analysis, respectively. The cells were cultured in 10% FBS/α-MEM, and once 80% confluence was achieved, the medium was immediately changed to osteogenic induction medium (10% FBS/α-MEM supplemented with 50 μg/mL ascorbic acid (Sigma-Aldrich), 20 nM dexamethasone and 8 mM β-glycerol phosphate, with or without 1, 5, or 10 μg/mL c-di-AMP (SML1231; Sigma-Aldrich). For signaling pathway investigations, cells were pretreated with 5 μM of the MyD88 inhibitor ST2825 (HY-50937; MedChemExpress, Monmouth Junction, USA), 10 μM of the ERK inhibitor U0126 (A1337; APEXBIO, Houston, USA), 15 μM of the p38 inhibitor SB203580 (A8254; APEXBIO), or 5 μM of the NF-κB inhibitor BAY11-7082 (S1523; Beyotime, Shanghai, China) for 3 h and then incubated with c-di-AMP. Four- and seven-day stimulations were used for ALP staining, qPCR and western blot analysis, and a 14-day induction was used for ARS staining.

For intracerebroventricular microinfusion, animals were placed in a stereotaxic apparatus (RWD Life Science Co., Ltd, 8001, Shenzhen, China) with the bregma used as the zero point. The left lateral cerebral ventricle was entered by using the following coordinates: anteroposterior, −0.8 mm; lateral, −1.5 mm and vertical, −4.0 mm. Anti-mouse/rat TNF-α antibody (dissolved in PBS; 250 ng/μL, 10 μL per rat; eBioscience™, 14-7423-85, San Diego, CA, USA), U0126 (dissolved in PBS/2% dimethyl sulfoxide, 5 μg/μL, 6 μL per rat; ApexBio Technology LLC, A1337, Houston, TX, USA) or the same volume of vehicle was microinfused into the left lateral cerebral ventricle 30 min before SAH. The infusions were performed at a rate of 0.5 μL/min using a microinfusion pump, and injection cannulas were left for an additional 2 min. Phorbol-12-myristate-13-acetate (PMA, dissolved in PBS/1% dimethyl sulfoxide, 100 μg/mL, 500 μg/kg; Sigma, P1585, St Louis, MO, USA) or the same volume of vehicle was injected intraperitoneally immediately after the microinjection of anti-TNF-α antibody.

L02 and HepG2 cells were cultured in Dulbecco’s minimal eagle medium containing 10% fetal bovine serum at 37°C in a 5% CO₂ humidified atmosphere. The in vitro model of NAFLD was induced by treatment with a mixture of 1 mM free fatty acids (FFA; oleic acid: palmitic acid = 2:1) containing 1% FFA-free bovine serum albumin (BSA) for 24 h as described by Gómez-Lechón et al.16 (link) Cells were transfected with RASAL2 overexpression plasmid (GV492-RASAL2: GeneChem, Shanghai, China; pNTAP-RASAL2: provided by Professor Wu Xiaohui of Fudan University) or siRNAs (siRASAL2: forward 5′-GCAGGACAGUUCAACCUAATT-3′; siTET1: forward 5′-CCUCCAGUAAUGGCUAUAATT-3′; Scrambled: forward 5′-UUCUCCGAACGUGUCACGUTT-3′; Biotend, Shanghai, China), followed by FFA treatment with or without AKT inhibitor triciribine and MEK inhibitor U0126 (both from Apexbio, Houston, TX, USA) for 24 h.