Wm 266 4

The metastatic human melanoma cell lines MeWo, C32, A2058, SK-Mel-28, SH-4, A375, Hs 852.T, the human erythroleukemia cell line K562, the B cell leukemia NALM-18, and the primary/metastatic pair WM-115/WM-266–4 (derived from the same patient) were purchased from the American Type Culture Collection (ATCC, Virginia, USA). The primary T1 and the metastatic melanoma G1 cell lines, derived from the same patient, were obtained from the Institute Gustave Roussy (Villejuif, France). The abovementioned cell lines were expanded in RPMI-1640 medium (Euroclone, Italy) supplemented with 2 mM L-glutamine (Euroclone), 1% streptomycin and penicillin (Euroclone), and 10% fetal bovine serum (FBS; Thermo-Fisher Scientific, Massachusetts, USA). In some experiments, melanoma cell lines were treated for 36 h with 1 ng/mL of interferon alpha (IFN-α) (Merck, Canada) and 1 ng/ml of anti-CD40 monoclonal antibodies (mAb) (clone 5C3 - Functional Grade, Thermo-Fisher Scientific). In other experiments, WM-115, WM-266–4, T1, G1, A2058, and SK-Mel-28 melanoma cells were treated with 100 ng/mL of phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich) for 3 h as previously described (28 (link)). All cell lines were confirmed as mycoplasma-negative by reverse transcription PCR (RT-PCR) (Eurofins, Luxembourg).

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).