Human tgf beta1

Resveratrol was purchased from Shanghai Standardization for the Traditional Research Center and dissolved in dimethyl sulfoxide (DMSO). Human TGF-beta1 was obtained from PeproTech (USA). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) was purchased from Promega (Madison, WI, USA). QuicBlock^TM Blocking Buffer for Immunol Staining, Antifade Mounting Medium with DAPI, QuicBlock^TM Secondary Antibody Dilution Buffer for Immunofluorescence and QuicBlock^TM Primary Antibody Dilution Buffer for Immunol Staining were purchased from Beyotime (Shanghai, China). Antibodies against MMP-2, MMP-9, α-SMA, Smad2, Smad3, P-Smad2, P-Smad3, Snail1, and Slug were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fibronectin, P-PI3K, PI3K, P-AKT, AKT, E-cadherin, and vimentin antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). LY290042, SB431542, SP600125, PDTC, and PD98059 were purchased from Selleck Chemical (Houston, TX, USA). PDTC and PD98059 were purchased from MedChemExpresss (New Jersey, NJ, USA). IRDye^TM fluorescence antibodies were obtained from Li-Cor Bioscience (Lincoln, NE, USA).

si-YTHDF1s and negative control siRNA-NC were acquired from Guangzhou Ribobio, China. The selected sequences of siRNAs were as follows: si-YTHDF1-1: 5′-GGAACAACATCTATCAGCA-3′, si-YTHDF1-2: 5′-GCTCAACCGCAGTATCAGA-3′, si-YTHDF1-3: 5′-GGAAACGTCCAGCCTAATT-3′. The establishment of sh-YTHDF1 cell line was co-transfect the vector pLKO.1 expressing sh-YTHDF1 with the packaging vectors psPAX and vesicular stomatitis virus-expressing envelope vector pMD2G in HEK293T cells for 72 h to produce lentiviral particles. After incubating lentivirus in tumor cells for 48 h with polybrene, 1 µg/ml puromycin (Biosharp, China) was used to screen stable cell lines. Cells treated with Human TGF-beta1 (PeproTech, 100-21-10) at 10 ng/ml for 48 h or treated with SC79 (MCE, HY-18749) at 4 µg/ml for 1 h. Cell transfections were conducted using Lipofectamine 3000 (Invitrogen, USA) according to the instructions and protocols.