Sk mel 24

WM115, RPMI7951, SKMEL24, WM793, SKMEL28, SKMEL5, A375, SKMEL3, and MALM3M human melanoma cell lines were purchased from ATCC. 501MEL and WM9 cell lines were kindly provided by Dr Robert Ballotti and M4BE cell line by Dr Thibault Voeltzel (Centre Léon Bérard). All these BRAF^V600 human melanoma cell lines were cultured in DMEM complemented with 10% FBS (Cambrex) and 100 U/ml penicillin–streptomycin (Invitrogen). In order to authenticate the cell lines, the expected major genetic alterations were verified by NGS sequencing. The absence of mycoplasma contamination was checked every 3 weeks with the MycoAlert detection kit (Lonza). Patient‐derived short‐term cultures (< 10) were established from BRAF^V600 metastatic melanomas, before treatment for GLO and C‐09.10, or after acquisition of resistance to vemurafenib for ESP and GOKA. C‐09.10 were kindly provided by Dr Robert Ballotti (Nice). These short‐term cell cultures were grown in RPMI complemented with 10% FBS and 100 U/ml penicillin–streptomycin. PLX4032/vemurafenib and GDC0973/cobimetinib were purchased from Selleck Chemicals (Houston, TX, USA) and reconstituted in DMSO. Generation of A375‐R and SKMEL5‐R resistant models was performed by treating cells chronically with increasing doses of PLX4032 for 2–3 months. All BRAFi‐resistant cell lines were permanently cultured in the presence of 3 μM PLX4032.

Ten human melanoma cell lines (A375, A375M, CHL-1, MEL224, MEL501, MEL505, MEWO, RPMI7951, SKMEL24, and SKMEL3) were kindly provided by Andreja Ambriović Ristov, PhD and Neda Slade, PhD. Cell lines HS895.SK (ATCC CRL-7636; Accession number CVCL_0992), HS895.T (ATCC CRL-7637; Accession number CVCL_0993), HS940.T (ATCC CRL-7691; Accession number CVCL_1038) and SKMEL2 (ATCC HTB-68; Accession number CVCL_0069) were purchased from the ATCC (Manassas, VA, USA). HS895.SK cell line represents a healthy control: skin keratinocytes isolated from the same patient as the HS895.T melanoma cell line. All cell lines were maintained in recommended medium: Dulbecco’s Modified Eagle Medium (Merck KgaA, Darmstadt, Germany), RPMI 1640 medium (Merck KgaA, Darmstadt, Germany), or Eagle’s Minimum Essential Medium (Merck KgaA, Darmstadt, Germany), supplemented with 10% FBS (Merck KGaA, Darmstadt, Germany), 1 mM sodium pyruvate, 1% streptomycin/penicillin and 4 mM L-glutamine (Gibco Thermo Fisher Scientific, Waltham, MA, USA).

The experiments were performed in accordance with the Declaration of Helsinki guidelines. The study was approved by the Clinical Research Ethics Board of the University of British Columbia (Certificate H12-02653). With informed consent, biopsies were obtained and stored in RNAlater solution (Life Labs) as previously described [23 (link), 31 (link)] [32 (link)]. Biopsy tissues were archived and stored in −20°C in RNAlater solution (Invitrogen, Canada). Human epidermal melanocytes were purchased from ScienCell (Carlsbad, USA) and cultured in melanocyte medium (2201, ScienCell, Carlsbad, USA). Melanoma cell lines A375, RPMI 7951, SH4, WM-115, SK-MEL-1, SK-MEL-3, SK-MEL-24 were purchased from ATCC (Manassas, USA). Cells were cultured in growth medium as Dulbecco's modified Eagle's medium (DMEM) (Hyclone, Logan, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, USA) and 1X Antibiotic-Antimycotic (15240062, Gibco, Burlington, Canada) at 37°C in 5% CO2 humidified atmosphere.

Human melanoma lines SK-MEL-1, A375, G-361, SK0MEL-3, SH-4 and SK-MEL-24 were purchased from ATCC. They were grown in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 50 mM l-glutamine at 5% CO₂/37 °C. Primary melanocyte cells were ordered from Lonza (adult donors). They were grown in Medium 254 (Thermo Fisher Scientific) supplemented with human melanocyte growth supplement (#S0025, Life Technologies) at 5% CO₂/37 °C.

The human epidermal melanocytes (HEMa-LP) and melanoma cell lines (A375, SK-MEL-24, WM451, WM35) were purchased from the American Type Culture Collection (Manassas, VA, USA) and incubated with DMEM medium containing 10% fetal bovine serum in an incubator at 37°C. When the cell confluence was over 80%, melanoma cells were seeded in 6-well plates and cultivated for 24 h before transfection. After that, melanoma cells were transfected with TRIM14 knockdown (siRNA-TRIM14) or overexpression (Oe-TRIM14) plasmid and their corresponding negative controls (siRNA-NC or Oe-TRIM14) for the indicated time, and then they were used for the following assays.

The murine D4M melanoma cell line was purchased from Kerafast (Boston, MA) [15 (link)]. The human melanoma cell lines A375 and SK-MEL-24 were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbecco’s modified Eagle medium (DMEM) in a humidified incubator at 37°C with 5% CO2. Two human CAF cell lines (224350P1/M50 and DT01027P1/M27) were isolated from surgically excised human melanoma tissues and obtained from Asterand bioscience (Detroit, MI). All culture media were supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) 10,000 U/ml penicillin and 10,000 U/ml streptomycin. All cell culture reagents were purchased from Thermo Fisher Scientific (Rochester, NY) unless otherwise stated. The isolation and maintenance of primary human fibroblasts were approved by the Institutional Review Board and the Institutional Biosafety Office of the University of Cincinnati. Experimental procedures involving biosafety issues were carried out under the University of Cincinnati Institutional Biosafety Committee protocol 16-08-17-01.