Sk mel 28

Malme-3M, Sk-Mel-2, Sk-Mel-5, Sk-Mel-28, M14 and Lox-IMVI melanoma cell lines were obtained from the Department of Developmental Therapeutics, National Cancer Institute (Bethesda, MD, USA). WM-115 and WM-266-4 melanoma cell lines were obtained from the European Association Culture Collection (UK). Malme-3M, Sk-Mel-2, Sk-Mel-5, Sk-Mel-28, M14 and Lox-IMVI cell lines were maintained at 37°C with 5% CO₂ in RPMI-1640 medium (Sigma-Aldrich, Co. Wicklow, Ireland) with 10% fetal calf serum (FCS; Lonza, Tewkesbury, UK). WM-115 and WM-266-4 were maintained at 37°C with 5% CO₂ in minimal essential medium (MEM; Sigma-Aldrich) with 10% FCS (BioWhittaker, Walkersville, MD, USA), 2 mM L-glutamine (Life Technologies, Dublin, Ireland), 1 mM non-essential amino acids (Life Technologies) and 1 mM sodium pyruvate (Life Technologies). Stock solutions of fascaplysin (Merck Millipore, Watford, UK) (10 mM), PLX4032 (Sequoia Research Products Ltd., Pangbourne, UK) (10 mM), elacridar (Sigma-Aldrich) (10 mM) and temozolomide (Sigma-Aldrich) (103 mM) were prepared in dimethyl sulfoxide (DMSO) PD0332991 (provided by Pfizer, Peapack, NJ, USA) (10 mM) was prepared in ultrapure water.

The human monocytic cell line THP-1 (TIB-202), and BRAF mutated human malignant melanoma cell lines SK-MEL-28 (HTB72) and MALME-3M (HTB64) were purchased from American Type Culture Collection (ATCC; Rockville, MD, USA). THP-1 monocytes were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Gibco™) medium supplemented with 10% fetal bovine serum (FBS) (Gibco™ Thermo Fisher Scientific, Inc., Waltham, MA, USA), 1% penicillin–streptomycin (P/S) (Gibco™), and 1% l-glutamine (Gibco™). BRAF mutated melanoma cells isolated from the excised tumors (MM-55) as well as SK-MEL-28 and MALME-3M were maintained in standard Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS, 1% penicillin–streptomycin (P/S), and 1% l-glutamine. MAFs were propagated in MAF medium (DMEM supplemented with 20% FBS, 1% penicillin–streptomycin (P/S) and 1% l-glutamine), and half of the medium was refreshed every other day.

Human melanoma cell lines A375, RPMI, and SK-MEL-28; human keratinocytes; and HaCaT were purchased from the American Type Culture Collection (ATCC) and human skin fibroblast, 1BR3 was obtained from the European Collection of Authenticated Cell Cultures (ECACC). A375 and HaCaT were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) high-glucose medium supplemented with 10% fetal bovine serum (FBS, Gibco, ThermoFisher Scientific Waltham, MA, USA) and 1% penicillin/Strep, 10,000 IU/mL, Sigma-Aldrich, St. Louis, MO, USA) and RPMI, SK-MEL-28 and 1BR3 were cultured in Eagle’s Minimum Essential Medium (EMEM, Sigma-Aldrich, St. Louis, MO, USA) high-glucose medium supplemented with 10% fetal bovine serum (FBS, Gibco, ThermoFisher Scientific, Waltham, MA, USA) and 1% penicillin/Strep, 10,000 IU/mL, Sigma-Aldrich, St. Louis, MO, USA). The cells were incubated under standard temperature conditions of 37 °C and humidity containing 5% CO₂.

Human melanoma cell lines: A375, RPMI, and SK-MEL-28 were purchased from the American Type Culture Collection (ATCC). A375 were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) high-glucose medium supplemented with 10% fetal bovine serum (FBS, Gibco, ThermoFisher Scientific, Waltham, MA, USA) and 1% penicillin/Strep, 10,000 IU/mL (Sigma-Aldrich), and RPMI and SK-MEL-28 were cultured in Eagle’s Minimum Essential Medium (EMEM, Sigma-Aldrich) high-glucose medium supplemented with 10% fetal bovine serum (FBS, Gibco, ThermoFisher Scientific) and 1% penicillin/Strep, 10,000 IU/mL (Sigma-Aldrich). The cells were incubated under standard temperature conditions of 37 °C and humidity containing 5% CO₂.

Human melanoma cell lines WM266‐4, A2058, and SK‐MEL‐28 were purchased from American Type Culture Collection (ATCC), and HUVECs were a kind gift from Dr. Annie Cheung. Purchased cell lines were tested to exclude mycoplasma contamination by the Centre for PanorOmic Sciences, Li Ka Shing Faculty of Medicine, HKU. Both WM266‐4 and SK‐MEL‐28 cells were cultured in 10% serum‐containing EMEM (Sigma), A2058 and human embryonic kidney 293T cells were cultured in 10% serum‐containing DMEM (Gibco), and HUVECs were cultured in Medium 200RPF (Gibco) with low serum growth supplement (ThermoFisher) according to the manufacturer's instruction. All cell lines were maintained in a 37 °C humidified incubator supplemented with 5% CO₂. The list of culture conditions for each cell line is shown in Table S5 (Supporting Information).
Plasmids encoding V5‐DEPDC1B, SOX10, SCUBE3, CDC16‐HA, and truncated CDC16 N1‐266‐HA and N262‐620‐HA were used. These genes were amplified from WM266‐4 cDNAs and cloned into pLVX‐IRES‐EF1a‐puro vector using restriction enzymes. The primer sequences and enzyme sites are listed in Table S6 (Supporting Information). A pLKO.1‐TRC vector was used to knockdown DEPDC1B, SOX10, SCUBE3, and CDC16. The shRNAs were generated by IDT and cloned into pLKO.1‐TRC at the AgeI and EcoR1 sites. The shRNA target sequences are listed in Table S7 (Supporting Information).