The present study used eight melanoma cell lines (listed in Table SI ). Their mutational status (BRAF and NRAS mutations) is shown in Table SII . The origin of cells has been described previously (31 (link),32 (link)). Cells were cultivated in RPMI medium (MilliporeSigma) supplemented with 10% FCS (Thermo Fisher Scientific, Inc.), glutamine and antibiotics (MilliporeSigma) at 37°C and 5% CO2 in 100% humidity. All cell lines were authenticated and tested for mycoplasma using a mycoplasma detection kit (MP0035; MilliporeSigma). Cells were passaged every 72–96 h using a trypsin-EDTA solution. When plated, cells (5×105) were seeded from a stable culture into 12-well plates and incubated for 24 h at 37°C prior to inhibitor treatment or transfection, unless specified otherwise. Normal human melanocytes were purchased from Cascade Biologics Inc. and cultivated according to the manufacturer´s instructions. The generation of inducible melanoma cell lines in which melanoma-associated transcription factor (MITF) protein can be downregulated by the addition of doxycycline (Tet-on system) has been previously described (32 (link)).
Normal human melanocytes
Manufactured by Thermo Fisher Scientific
Sourced in United States
Normal human melanocytes are primary cells derived from the epidermis of normal human skin. They are specialized pigment-producing cells responsible for skin coloration.
Automatically generated - may contain errors
Lab products found in correlation
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Glutamine, by Merck Group (1 mentions)
Rpmi medium, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (1 mentions)
Sk mel 5, by Korean Cell Line Bank (1 mentions)
Sk mel 2, by Korean Cell Line Bank (1 mentions)
Endo h, by New England Biolabs (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
M254 medium, by Thermo Fisher Scientific (1 mentions)
Human melanocyte growth supplement, by Thermo Fisher Scientific (1 mentions)
Gw4869, by Santa Cruz Biotechnology (1 mentions)
3 protocols using normal human melanocytes
1
Melanoma Cell Line Cultivation and Characterization
2
Detailed Melanoma Cell Culture Protocol
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The human melanoma MNT-1 cell line was kindly provided by Dr. Ai-Young Lee at Dongguk University, who originally received it as a gift from Dr. Vincent J. Hearing at National Institutes of Health, Bethesda, Maryland, USA [42 (link)]. The cells were cultured at 37°C in MEM (Gibco, Carlsbad, CA, USA) containing 20% FBS, 10% DMEM (Lonza, Basel, Switzerland), 20 mM HEPES and antibiotics. The WM266-4 human melanoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The SK-MEL-1, SK-MEL-2, SK-MEL-3, and SK-MEL-5 human melanoma cell lines were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea). Those cells were maintained at 37°C in RPMI1640 medium (Gibco) containing 10% FBS and antibiotics. Normal human melanocytes (Cascade Biologics, Portland, OR, USA) were cultured in M254 medium (Cascade) containing human melanocyte growth supplement-2 (Cascade). Endo H and PNGase F were purchased from New England Biolabs (Hitchin, UK).
3
Culturing Normal Human Melanocytes
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Normal human melanocytes (Cascade Biologics, Portland, OR) were maintained in M-254 medium (Cascade Biologics) containing human melanocyte growth supplement (Cascade Biologics). GW4869 (Santa Cruz Biotechnology, Santa Cruz, CA), pifithrin-m (Sigma, St.
Louis, MO), and b-estradiol (estrogen) (Sigma) were dissolved in DMSO. The FN inhibitor tetrapeptide H-Arg-Gly-Asp-Ser-OH (Santa Cruz Biotechnology) was dissolved in phosphate buffered saline (PBS).
Louis, MO), and b-estradiol (estrogen) (Sigma) were dissolved in DMSO. The FN inhibitor tetrapeptide H-Arg-Gly-Asp-Ser-OH (Santa Cruz Biotechnology) was dissolved in phosphate buffered saline (PBS).
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