T 5224

To evaluate the cell response to different drugs, cell proliferation was determined by the Cell Counting Kit-8 (7sea biotech, China) assay after treatment. In brief, HL-60 and THP-1 cells (5 × 10⁴/ml) were seeded in 96-well culture plates and incubated with T-5224 (Selleck, USA) (concentration gradient is set to 0, 10, 20, 40, 60, and 80 μM) or united Ara-C (SinoPharm YiXin, China) (concentration gradient is set to 0.625, 1.25, 2.5, 5, 10, and 20 μM) treatment for a period of time (24, 48, and 72 h). After certain times, 10 μl of CCK8 solution was added to each well for a 3-h culture at 37°C. Absorbance was measured by a spectrophotometer (Bio Tek Instruments, USA) at a wavelength of 450 and 630 nm. The calculation formula of relative cell vitality (%) is: (experimental well - blank well)/(control well - blank well) × 100%.

HFF-1 cells were seeded in 6-well plates with DMEM at 200,000 cells/well. Cells were treated with control (DMEM with 2% FBS-added vehicle) or 4 ng/mL TGF-β1 (R&D Systems) with or without each inhibitor, and the lysates were collected after 48 h for quantification by WB. The following inhibitors were used: LY364947 (123-05981, Fujifilm), SB431542 (dispensed from a Tocriscreen Kinase Inhibitor Toolbox [3514, Tocris Bioscience]), and T-5224 (S28966, Selleck).

Antibodies against Collagen type I, α-SMA, SMAD2, SMAD3 were purchased from Cell Signaling Technology (MA, USA). Antibodies against p-SMAD2, p-SMAD3 and p-SMAD2/3 were obtained from Boster Biological Technology Co., ltd (Wuhan, China). Antibodies against SRPX2, β-ACTIN, SMAD7 and FIBRONECTIN were acquired from Proteintech Group, Inc (IL, USA). Cholesterol and DSPC were acquired from Sigma-Aldrich, Inc. (St. Louis, MO) and mPEG2000-DMG (MW2660) was purchased from NOF Co., Ltd. (Kawasaki Japan). The cationic lipidoid C12-200 was generated through ring opening of epoxides by amine substrates with a previously reported method 16 (link). The selective inhibitor of p-SMAD3 (SIS3-HCL), the selective inhibitor of TGF-β Receptor type I receptor (SB-431542) and Recombinant Human TGF-β1 were obtained from MedChemExpress (NJ, USA). T-5224, a selective inhibitor targeting AP-1, was obtained from Selleck Chemicals (Texas, USA). BLM was obtained from Hisun Pharmaceutical Co., Ltd (Zhejiang, China). RT-PCR assay kit was supplied by Takara (Liaoning, China). A hydroxyproline assay kit was obtained from BioVision (CA, USA).

Cells were plated subconfluently in 96-well plates, then serum starved and treated with the following inhibitors: vismodegib (GDC-0449; SelleckChem), CCG-1423 (Cayman Chemicals), T-5224 (SelleckChem), SR11302 (Cayman Chemicals), SB431542 (SelleckChem), SP600-125 (SelleckChem), JNK-IN-8 (SelleckChem), or RHO inhibitor I (Cytoskeleton Inc). Cell viability was measured after 72-h treatment using CellTiter 96 Aqueous One Solution (Promega) and normalized to DMSO vehicle control. Each condition was tested six times. For each drug and cell line, the half-maximal inhibitory concentration was determined as the drug concentration giving the half-maximal response compared with the control (DMSO-treated) conditions.