Tgf β1 recombinant protein

The endometrial tissue samples used for the primary culture were removed and transported immediately to the laboratory. They were chopped to a size of 1 mm³ and washed with PBS three times. After the cells were fully digested with trypsin/EDTA (TBD Science, Tianjin, China) in a humidified atmosphere of 5% CO₂ at 37°C, the cells were pelleted by centrifugation for 5 min, vigorously resuspended in RPMI 1640 (Gibco; Shanghai, China) supplemented with 10% fetal bovine serum (TBD Science, Tianjin, China) and 100 U/ml penicillin (Gibco, Shanghai, China) and 100 μg/ml streptomycin (Gibco, Shanghai, China), plated, and allowed to settle for up to 3 days. All the cells were identified to be epithelial cells and interstitial cells using immunofluorescence. The endometrial cells were exposed to 1% O₂ hypoxia in the presence or absence of 10 ng/ml TGF-β1 recombinant protein (Peprotech, NJ, USA) or 10 μmol/L of the TGF-β1 signal pathway inhibitor galunisertib (LY2157299) (Selleck Chemicals, Houston, USA). Hypoxic conditions were maintained using a modular incubator chamber (Hinasama, Tokyo, Japan) with 5% CO₂ and 1% O₂ balanced with N₂ gas. Data from cells cultured in 21% O₂ were used as normal controls.

Human pancreatic cancer cell lines (BxPC-3, PANC-1, and CFPAC-1) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA). Pancreatic cancer cells were serum-starved for 12 h before treatment with 10 µM LPA (Sigma-Aldrich) for another 24 h and tested for gene expression, migration, and invasion. TGF-β1 recombinant protein (50 ng/mL; PeproTech, Rocky Hill, NJ, USA) was also used to induce pancreatic cancer cell migration and invasion. In some experiments, pancreatic cancer cells were treated with LY2090314 (50 nM; Selleckchem, Houston, TX, USA) for 24 h before transfection with CMTM8 shRNA.