Anti mmp1

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).

Alantolactone (HY-N0038) was obtained from MedChem Express (MCE). Recombinant mouse IL-1β cytokine was provided from R&D Systems (501-RL-010, United States). Solarbio supplied safranin O liquor (Beijing, China). Primary antibodies applied in this research were: anti-iNOS, anti-MMP-13 which were acquired from Abcam (Shanghai, China), anti-COX2, anti-LC3Ⅱ/Ⅰ, anti-P-STAT3, anti-P-P65, anti-P-IκB, anti-P/T-mTOR which were purchased from CST (Beverly, MA, United States), anti-STAT3, anti-MMP-1, anti-MMP-3, anti-ATG5, anti-P62, anti-P65, anti-IκB, anti-P/T-PI3K, anti-P/T-AKT, anti-GAPDH which were afforded from Proteintech Group (Wuhan, Hubei, China) and anti-ADAMTS5 which was supplied from Boster Biological Technology (Wuhan, Hubei, China). Secondary antibodies, Cy3 and FITC Conjugated AffiniPure Goat Anti-Rabbit IgG, DAPI staining solution, collagenase type II and trypsin were got from Boster Biological Technology (Wuhan, Hubei, China). HanBio Inc. (Shanghai, China) served the mRFP-GFP-LC3 adenoviral vectors.

Total cellular protein lysates were prepared by harvesting the cells in a protein extraction buffer for 1 h at 4°C as described previously [24 (link)]. GAPDH expression was used as the protein loading control. Anti-Akt, phospho-Akt, phospho-Smad2 phospho-p70S6 K, anti-TGF-β, anti-Beclin 1 and anti-LC3 antibodies were obtained from Cell Signaling Technology (Ipswich, MA, USA). Anti-MMP-1, anti-MMP-9, anti-procollagen type 1 and anti-GAPDH antibodies were obtained from Proteintech (Rosemont, IL, USA); anti-Samd2/3 antibody was obtained from Santa Cruz (Dallas, TX, USA); and anti-p70S6 K antibody was obtained from Abcam (Cambridge, MA, USA).

Equal amount of protein from each group was fractionated by 8% or 10% sodium dodecylsulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane (Millipore, Germany). Membranes were incubated with primary antibody overnight at 4°C, followed by horse radish peroxidase (HRP)-conjugated secondary antibodies. The antibodies used in this study were anti-integrin β1, anti-pFAK-Y397 (1:1000, Abcam), anti-FAK, anti-AKT, anti-pAKT-Ser473 (1:1000, CST), anti-MMP-1(1; 1000, proteintech, USA) and anti-β-actin (1:3000, proteintech). Proteins were detected using Clarity™ western ECL substrate (Bio-Rad Laboratories) and viewed by the imaging system (Universal hood III, Bio-Rad).