The normal human colon mucosal epithelial cell line (NCM460), the low invasive human colon adenocarcinoma cell line (HT-29), the highly invasive human colon cancer cells (HCT-8), HCT-8/V and Bel7402 hepatocarcinoma cell line were purchased from KeyGEN BioTECH (Nanjing, China). All the cell lines were verified through short tandem repeat analysis. These cell lines were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) (Hyclone, USA) in a humidified atmosphere of 5% CO2 at 37°C (Supplementary Table 1 ). The HCT-8/V cell line was preserved in 1.6 mg/L vincristine.
The cells (1×106) were planted in a 60 mm dish. After the cells were incubated overnight, they were transfected with pcDNA3-Twist1, pcDNA3-negative, Twist1 siRNA, and control siRNA in serum-free Opti-MEM medium (Santa Cruz, California, USA) using Lipofectamine 2000 (Invitrogen, California, USA) according to the manufacturer’s instructions. The cells were collected after 48 h for the other experiments. To test for rescue of Twist 1 silencing, pcDNA3-ABCB1 vectors were co-transfected together with the Twist1 siRNA. For EMT induction, the cells were treated with 3 ng/mL of rh-TGF-β1 for 96 hours. The neutralizing TGF-β1 antibody was used to inhibit the TGF--β1 activity.
The cells (1×106) were planted in a 60 mm dish. After the cells were incubated overnight, they were transfected with pcDNA3-Twist1, pcDNA3-negative, Twist1 siRNA, and control siRNA in serum-free Opti-MEM medium (Santa Cruz, California, USA) using Lipofectamine 2000 (Invitrogen, California, USA) according to the manufacturer’s instructions. The cells were collected after 48 h for the other experiments. To test for rescue of Twist 1 silencing, pcDNA3-ABCB1 vectors were co-transfected together with the Twist1 siRNA. For EMT induction, the cells were treated with 3 ng/mL of rh-TGF-β1 for 96 hours. The neutralizing TGF-β1 antibody was used to inhibit the TGF--β1 activity.