Ipc298

Human melanoma cell lines such as IGR37, IGR39, and IPC‐298 were purchased from DSMZ; MEWO cell line was purchased from ICLC; SK‐MEL‐5 and SK‐MEL‐28 were purchased from NCI‐60; WM266.4 was purchased from ATCC; WM115, A‐375 and C32 were purchased from IZSBS; and HEK293T cells were purchased from ICLC.
All the cells were cultured in Dulbecco modified Eagle's medium (DMEM)+ 10% FBS S.A.+ 2 mM l‐Glutamine except for IPC‐298 that was cultured in RPMI‐1640+ 10% FBS S.A.+ 2 mM l‐Glutamine. Cell lines were tested for mycoplasma by mycoplasma PCR Test Kit.

A375 melanoma cells were purchased from ATCC, MelJuso and IPC298 cells were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) while the 501Mel melanoma cell line was obtained from Ruth Halaban (Dermatology department, Yale School of Medicine, New Haven, CT, USA). Normal Human Dermal Fibroblasts (NHDFs) were obtained from Heike Hermanns (University Hospital Würzburg, Würzburg, Germany). All melanoma cell lines were cultured in RPMI + Glutamax (Lonza BioWhittaker, Basel, Switzerland) + 10% FBS and 1% PS (10,000 U/mL Penicillin and 10,000 U/mL Streptomycin, Lonza BioWhittaker, Basel, Switzerland) while NHDFs were cultured in DMEM + 10% FBS and 1% PS (10,000 U/mL Penicillin and 10,000 U/mL Streptomycin (PS, Lonza BioWhittaker, Basel, Switzerland). All cells were grown at 37 °C in a humidified atmosphere at 5% CO₂. Cells were regularly tested to be mycoplasma free. Hypoxia treatment was performed at 37°C in a water-saturated atmosphere at 5% CO₂ in a hypoxia station (Invivo₂ 400, Ruskinn Technology Ltd, Bridgend, UK) at 1% O₂.

Cell lines DU145 (ACC 261), HEK293 (ACC 305), HeLa (ACC 57), IPC298 (ACC 251), IGR39 (ACC 239), IMR32 (ACC 165), and SH-SY5Y (ACC 209) were purchased from DSMZ (Braunschweig, Germany). MDA-MB-435S (HTB129) cells were from ATCC (Manassas, VA), PNT2 cells (ECACC95012613) were obtained from ECACC (Salisbury, UK). GL15 cells were kindly provided by Dr. Fioretti (University of Perugia, Italy). Each cell line was cultured in their respective recommended medium supplemented with 10% FCS (PAA Laboratories) at 37 °C in humidified 5% CO₂ atmosphere. For stablly transfected cell lines (HEK expressing K_V10.1 in the pTracerCMV vector, a cell line routinely used in our laboratory (4 (link), 6 (link)– (link)8 (link)), the selection compound Zeocin (Calya) was added to the culture medium at 3 μg/ml. Transient transfections were performed using FuGENE (Roche Applied Science) or Lipofectamine 2000 (Invitrogen). Proliferation was estimated using Alamar Blue (BIOSOURCE) or WST assays (Roche Applied Science) as described (50 (link)) or by live cell imaging in an IncuCyte Zoom system (Essen Biosciences) to determine the percent confluence as a function of time.