Fbs premium heat inactivated

Melanoma cell cultures were established from surplus cerebral melanoma metastases after having obtained written, informed consent approved by the local IRB (EK647 and EK800). Cells were grown in RPMI (Sigma RPMI-1640, #R0883) supplemented with 5 mM l-glutamine (gibco l-glutamine, #25030), 1 mM sodium pyruvate (Sigma sodium pyruvate, #S8636) and 10% FBS (PAN biotech FBS Premium heat inactivated, #P30-1902, Aidenbach, Germany). Culture medium was changed every 2–3 days to ensure optimum conditions of growth, using aseptic techniques and a laminar flow bench. Cells were tested for mycoplasma contamination (Invivogen PlasmoTest Mycoplasma Detection Kit, #rep-pt) prior to their use for the described techniques.

The BRAFV600E mutated primary human melanoma cell line M000921 has been established from surplus material from cutaneous melanoma metastases. This cell line has been previously characterized as a proliferative-phenotype melanoma culture (by means of melanoma phenotype-switching model) and shared with multiple studies and international laboratories [24 (link), 47 (link)–50 (link)]. Expression data from a Affymetrix HG-U133 plus 2.0 oligonucleotide microarray is deposited on GEO (GSM700745). Written informed consent was approved by the local IRB (EK647 and EK800). Clinical diagnosis was confirmed by histology and immunohistochemistry. Melanoma cell culture was grown in RPMI (Sigma RPMI-1640, #R0883) including 5 mM l-glutamine (gibco l-glutamine, #25030), 1 mM sodium pyruvate (Sigma sodium pyruvate, #S8636) and 10% FBS (PAN biotech FBS Premium heat inactivated, #P30-1902, Aidenbach, Germany). M000921 melanoma cells were kept in medium containing 5 ng/ml human recombinant TGFβ (R&D Systems, #240-B) for a period of 12 days. Medium containing fresh TGFβ protein was changed every 3 days. RNA was extracted from both untreated and TGFβ treated cell culture using TRIzol reagent (Invitrogen, USA) following manufacturer’s protocol. RNA labelling, hybridization to microarray (HG-U133 plus 2.0, Affymetrix) and data were statistically analyzed as described previously [49 (link)].