Hema lp

Human epidermal melanocytes HEMa-LP (C0245C) were provided by Thermo Fisher Scientific (Waltham, MA, USA). Four human melanoma cell lines A375 (CRL-1619), M21 (BAA-1539), B16F10 (CRL-6475) and SK-MEL-2 (HTB-68), were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). All cell lines were cultured at 37 °C with 5% CO₂. Then, cells were all maintained continuously in DMEM medium (Thermo Fisher Scientific) supplied with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% antibiotics (Invitrogen, Carlsbad, CA, USA).

Human melanoma cell lines SKMEL28, A375 were obtained from ATCC, cultured and maintained in RPMI 1640 supplemented with 10% of heat-inactivated fetal calf serum (FCS) (endotoxin level < 0.1 EU/ml), 50 U/ml penicillin/streptomycin and 2 mM L-glutamine (Euroclone, Milan, Italy) at 37 °C in a humidified atmosphere containing 5% of CO₂ and 95% of air. Adult lightly pigmented human epidermal melanocytes (HEMa-LP) were cultured and maintained according to the manufacturer's instructions (ThermoFisher, Waltham, MA, USA). In tissue culture plates, cells were seeded at a confluence of 10–20%. Fresh serum-free medium was added to the cell culture once the cells reached 85–90% confluence. Conditioned medium (CM) was harvested after 24 h, filtered (0.20 µm pore size filter) and stored at – 20 °C [15 (link)]. In all the performed experiments, control medium, melanoma-derived CM as well as the medium in which neutrophils were cultured, was supplemented with 5% FCS. Mycoplasma was routinely screened in all cell lines by PCR (Merk, Darmstadt, Germany).

The human SOX9 cDNA was cloned into the lentiviral pWPI vector (Addgene plasmid 12,254). The human NEDD9 cDNA fragment was amplified using pEF-HEF1 as a template and cloned into lentiviral vector pLVX-EF1α-puro (Clontech). The shRNA against the human SOX10 (5’-GACTTCGGCAACGTGGACATT-3′) and NEDD9 (5’-GAGACACCATCTACCAAGTTT-3′) were designed based on the principles from The RNAi Consortium (https://www.broadinstitute.org/rnai/public/) and cloned into lentiviral vector pLKO.1-puro. pLKO.1-TRC control was gift from David Root (Addgene plasmid #10879).
Human epidermal melanocyte (HEMa-LP) was purchased from ThermoFisher and cultured in Medium-254 supplemented with HMGS-2. Human melanoma cell lines A375M, UACC-457, UACC-827, UACC-903 except SK-MEL-28 and human embryonic kidney cell line 293 T were cultured in DMEM medium with high glucose (Life Technologies) supplemented with 10% fetal bovine serum (FBS) (BioSera) and 100 U/ml penicillin-streptomycin (Life Technologies). RPMI-1640 medium (ThermoFisher) was used to culture Me300 kindly provided by D Leung, the Hong Kong University of Science and Technology and SK-MEL-28. Human melanoma cell line WM266–4 was obtained from ATCC and cultured in EMEM medium (Sigma) supplemented with 10% FBS and 100 U/ml penicillin-streptomycin. Cell lines were authenticated by cell profiling (AmpFISTR Identifier PCR Amplification kit, Life Technologies).

The melanoma cell lines SK-MEL-28 and A375 were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). The melanoma cell lines M21 and SK-MEL-110 were kindly provided by the Central Laboratory, Tongji University Affiliated People's 10^th Hospital. The normal human epidermal melanin cell line, HEMa-LP, was purchased from Thermo Fisher Scientific (Shanghai, China). All cells were cultured in high glucose Dulbecco's Modified Eagle's medium (DMEM; Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (FBS; Gibco-BRL Life Technologies, Paisley, UK) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified incubator with 5% CO₂.

Human melanoma cell lines A375, SKMEL2, and SKMEL28 were obtained from the ATCC. Their identity was verified by ATCC, and they were further were regularly authenticated based on their distinct morphology. These human melanoma cell lines are not reported in the database of commonly misidentified cell lines maintained by ICLAC. They were grown in DMEM medium (Life Technologies) supplemented with 10% heat-inactivated FBS (Atlanta Biologicals), 1X GlutaMAX (Life Technologies) and 1% Penicillin-Streptomycin (Life Technologies), and grown at 37°C, 5% CO₂, and periodically checked for mycoplasma using the Universal Mycoplasma Detection Kit (ATCC). Primary Human Epidermal Melanocytes from an adult lightly pigmented donor, (HEMa-LP) were obtained from ThermoFisher Scientific and cultured according to manufacturer instructions in Medium 254 supplemented with Human Melanocyte Growth Supplement-2.