Chl 1

Human melanoma cell lines CHL-1 and SK-MEL-2 were obtained from Cell Resource Center, Chinese Academy of Sciences. CHL-1 and SK-MEL-2 cells were cultured in DMEM (Gibco) and MEM (Gibco) medium, respectively, supplemented with 10% fetal bovine serum (Gibco) at 37 °C with 5% CO₂. Cell lines used in this study were authenticated by STR profiling and routinely tested as mycoplasma-free.

Five human melanoma cells lines, including A-375, 1205Lu, UACC903, CHL-1 and sk-mel-5, were purchased from American Type Culture Collection. Cells were maintained in RPMI-1640 medium (A-375, 1205Lu) or DMEM medium (UACC903, CHL-1 and sk-mel-5) respectively supplemented with 10% fetal bovine serum (Gibco, CA, USA) in a 37°C humidified atmosphere of 5% CO₂.
Knockdown of BANCR was induced by transfection with BANCR shRNA (Genepharma, Shanghai, China) using Lipofectamine2000 (Invitrogen, CA, USA). The target sequences were as follows: shRNA #1: 5′-CGGAAATAGACTGCAGCAC CAA-3′; shRNA #2: 5′-CCTTTATGGATTCAACTGTAAT-3′. Ectopic expression of BANCR was achieved through pCDNA3.1-BANCR transfection using Lipofectamine2000. Oligonucleotides for amplification of BANCR were as follows: sense: 5′- CAGGAAGGGGTGAATGAAGA-3′; antisense: 5′- CCAGTGCAGGGTAATGTGTG-3′.
Stable transfectants were selected in medium containing 600 µg/mL G418 (Invitrogen, CA, USA) and used in further assays or RNA/protein extraction.
For treatment with pharmacological inhibitors, the overnight starved cells were kept in complete medium containing 10 µM U0126 (inhibitor of the upstream ERK1/2 activator MEK1/2, Sigma-Aldrich, MO, USA) or 20 µM SP600125 (JNK inhibitor, Sigma-Aldrich, MO, USA). DMSO was used as a control. Medium containing inhibitor was renewed every day.