Trypan blue edta

Triton X-100, Tris-HCl, puromycin, Trypan blue EDTA, ribonuclease-A, and dimethyl sulfoxide (DMSO) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The anti-snail antibody was purchased from GenScript. The anti-p-snail and anti-Hsp27 antibodies were purchased from Abcam. Antibodies against IGFBP2, β-catenin, vimentin, and GAPDH were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Hep-J5 and Mahlavu cells were gifts from Dr. C. S. Yang, National Taiwan University, and Dr. C. P. Hu, Veterans General Hospital, Taiwan [20 (link), 36 (link), 37 (link)]. The HCC cell lines were grown in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL, Grand Island, NY, USA) supplemented with 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 10% fetal calf serum (FCS; Gibco BRL) and 2% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cells were maintained in a 5% CO₂ humidified incubator at 37°C as previously described [38 (link), 39 (link)].

Human OS cells MG63 were purchased from ATCC and cultured in Eagle's minimum essential medium (MEM) (Gibco BRL, Grand Island, NY, USA) containing 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 10% fetal calf serum (Gibco BRL, Grand Island, NY, USA), and 2% penicillin-streptomycin (10,000 U/mL penicillin and 10 mg/mL streptomycin). The 143B cell line was provided by Dr. Pei-Ni Chen (Chung-Shan Medical University) and cultured in RPMI supplemented with 10% fetal bovine serum. Cells were incubated in a humidified incubator (37°C, 5% CO₂) and were either subcultured or used before they reached 80% confluence. Triton X-100, Tris-HCl, neomycin, trypan blue/EDTA, ribonuclease-A, and dimethyl sulfoxide (DMSO) were obtained from Sigma Chemical Co. (St. Louis, MO). Antibodies against GRP94 and GAPDH were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Caspase 3, caspase 7, and PARP antibodies were purchased from Cell Signaling Technology (Danvers, MA, United States).