Colo320

The hepatocellular carcinoma cell line HepG2 and the CRC cell lines HCT116, Colo205, Colo320, and LoVo, were purchased from the RIKEN BioResource Center (Ibaraki, Japan). DLD-1 cells were kindly provided by the Cell Response Center for Biochemical Research Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). HCT15 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). DLD-1, HCT116, HCT15, Colo205, and Colo320 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS). HepG2 cells were grown in Dulbecco's Modified Eagle Medium (DMEM; Gibco) supplemented with 10% FBS. LoVo cells were grown in L-15 medium (Gibco) supplemented with 10% FBS. Mycoplasma contamination was not tested because neither we, nor other researchers in our institute, have encountered mycoplasma contamination over the past 4 years.

Human CRC cell lines Caco-2, HT-29, Colo-320, Colo-205, and DLD-1, and a normal colonic endothelial cell line CCD-18Co were all purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Caco-2, HT-29, and CCD-18Co cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Gaithersburg, MD, USA). DLD-1, HT-29, Colo-320, and Colo-205 were grown in Roswell Park Memorial Institute (RPMI; Gibco-BRL) medium. Both of the media were supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco-BRL). The cells were maintained at 37°C in a 5% CO₂ humid atmosphere.
For EMT induction, the cells were treated with transforming growth factor-β1 (TGF-β1) (10 ng/ml) for 24 h following serum starvation for 8 h.
LY294002 (20 μM) and AZD8055 (0.8 nM) both purchased from Sigma-Aldrich (St. Louis, MO, USA) were used as inhibitors of the PI3K/AKT and mTOR pathways, respectively.