Anti timp 2

PSCs or HP-1 cells were cultured for 24 h in the absence or presence of stimulators respectively. These cells were lysed using RIPA lysis buffer (Beyotime, China) containing protease inhibitor cocktail (Sigma, USA). Protein samples (15 μg each) from PSCs or HP-1 cells were separated on 10% SDS-PAGE gels, and transferred to nitrocellulose. After washing with Tris buffer saline containing 0.1% Tween 20 (TBS/T) and blocking with 2.5% non-fat milk, the membranes were separately incubated at 4℃ overnight with rabbit polyclonal anti-α-SMA, anti-CTGF, anti-Col1, anti-MMP-1, anti-MMP-2, anti-TIMP-2, anti-GAPDH antibodies (ProteinTech, USA). Blots were incubated for 1 h with HRP-linked goat anti-rabbit IgG (Beyotime), and washed extensively with TBS/T before detection using the ECL system (ThermoFisher Scientific, USA).

Apatinib mesylate tablets (AITAN®; 425 mg/tablet) were purchased from Hengrui Medicine Company (Jiangsu, China). Tablets were dissolved in 100% dimethyl sulfoxide (DMSO; MP Biomedicals, Santa Ana, CA, USA) to a concentration of 4 mmol/L and stored at –20°C. The stock solution was diluted to a working concentration with DMEM (Gibco, Grand Island, NY, USA), and the final DMSO concentration in cell culture was less than 0.1%. The BCA kit was purchased from Google Biotechnology Ltd. (Wuhan, China). The RNA extraction reagent TRIzol and the PCR reagent Power SYBR® Green PCR Master Mix were purchased from Life Technologies (Grand Island, NY, USA), and the PCR primers were synthesized by Google Biotechnology Ltd. (Wuhan, China). Anti-MMP-1, anti-MMP-2, anti-MMP-3, anti-MMP-7, anti-MMP-9, anti-TIMP-1, anti-TIMP-2, and anti-TIMP4 antibodies were purchased from Proteintech Group (Chicago, USA), and Anti-MMP-10, Anti-MMP-11, and Anti-MMP-16 antibodies were purchased from Biorbyt Ltd. (Cambridge, UK). Anti-TIMP-3 was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). An NF-κB pathway sampler kit (9936T) was purchased from Cell Signaling Technology Inc. (Danvers, MA, USA).