Dulbecco s modified eagle medium dmem

Cells were purchased from ATCC (Manassas, VA, USA) and were regularly tested for mycoplasma contamination. MCF-7 cells were maintained at 37 °C, 5% CO₂ in Alpha MEM (Wisent 310–011 (St-Bruno, QC, Canada)) supplemented with 10% fetal bovine serum (FBS) (Sigma F1051), 1% l-glutamine (Wisent 609–065), and 1% penicillin–streptomycin (Wisent 450–201). SK-BR-3 and HEK-293 cells were maintained at 37 °C, 5% CO₂ in Dulbecco's modified eagle medium (DMEM) (Wisent 319–005) supplemented with 10% FBS and 1% penicillin–streptomycin. Three days before experiments, cells were switched to phenol red-free DMEM (Wisent 319–050) containing charcoal-stripped FBS, 2% l-glutamine and 1% penicillin–streptomycin.

Four established canine melanoma cell lines were used: CML-1, CML-6 M, and CML-10c2 (originally described by Lauren Wolfe et al. from Auburn University), and 17CM98 from the University of Wisconsin. All cell lines were generously provided by Mike Huelsmeyer from the University of Wisconsin, Madison, Wisconsin. CML-1, CML-6 M, CML-10c2, and 17CM98 originated from an oral melanoma, a lymph node metastasis from a primary cutaneous melanoma, a primary cutaneous melanoma, and a lymph node metastasis from a primary oral melanoma, respectively [44 (link), 45 (link)]. Cells were grown in monolayer culture in Dulbecco’s Modified Eagle Medium (DMEM) (Wisent, St-Bruno, Quebec) with 10% fetal bovine serum (FBS) (Wisent, St-Bruno, Quebec), 100 U/mL penicillin/streptomycin and 2.50 μg/mL amphotericin-B (Thermo Fisher Scientific, Waltham, Massachusetts) added to the media. Cell cultures were kept in a controlled environment at 37 °C humidified air and 5% CO₂.

Dulbecco's modified Eagle medium (DMEM), penicillin-streptomycin, HEPES and fetal bovine serum (FBS) were purchased from Wisent (St. Bruno, QC, Canada). GlutaMax was from Life Technologies (Burlington, ON, Canada). ATP, suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) were from Sigma-Aldrich (Oakville, ON, Canada). The MEK1/2 (UO126), p38 MAPK (SB203580), PI3K (LY294002) and NF- κB (BAY-11-7082) inhibitors as well as 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were acquired from Calbiochem (Mississauga, ON, Canada). Dimethyl sulfoxide (DMSO) was from Fisher Scientific (Ottawa, ON, Canada). The rabbit polyclonal antibodies against MRP2 and β-tubulin were purchased from Cell Signaling Technology (Pickering, ON, Canada). Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG was from Santa Cruz (Santa Cruz, CA, USA) and the ECL reagent from Millipore (Toronto, ON, Canada). The cytotoxic drugs etoposide, cisplatin and doxorubicin were purchased from the chemotherapy pharmacy at the Université de Sherbrooke Hospital Center (Sherbrooke, QC, Canada).

HEK293T and HT1080 cells (ATCC) were cultured in Dulbecco’s modified Eagle medium (DMEM; Wisent) supplemented with 10% fetal bovine serum (Sigma), 1 U/ml penicillin, 1 μg/ml streptomycin, and 3 μg/ml glutamine (Corning) at 37°C and 5% CO₂. Antibodies against NPC1 (Abcam), Flag (Sigma), mCherry (Abcam), LC3 (Novus Biologicals), GAPDH (Abcam), and vinculin (Abcam) were used.

MDA-MB-453, MCF-7, BT474, ZR-75-1, MDA-MB-231, HCC1806, and MCF-10A were purchased from American Type Culture Collection (ATCC), while SUM1315 was friendly and generously obtained from Stephen Ethier (University of Michigan, AnnArbor, MI, USA). MCF-10A, MDA-MB-453, MCF-7, BT474, ZR-75-1, MDA-MB-231 and SUM1315 cells lines were cultured in Dulbecco’s modified eagle medium (DMEM) (Wisent, China), and HCC1806 cell line was cultured in RPMI 1640 (Wisent, China). 10% fetal bovine serum, 100 mg/ml streptomycin and 100 U/ml penicillin were added into DMEM or RPMI 1640.

HEK293T and HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Wisent Inc.) supplemented with 10% fetal bovine serum and MDCK II cells were maintained in DMEM supplemented with 5% FBS. For plasmid transfection, Lipofectamine 2000 reagent (Invitrogen) was used according to the manufacturer’s instructions. Cells were cultured at 37 °C for 30–48 h before lysis. In order to generate stably transfected HEK293T cell lines, cells were transfected using Lipofectamine 2000 (Invitrogen) with 2 µg of FLAG-EPB41L5 cDNA or control pFLAG empty vector. After selection with 2 μg/ml of Puromycin for two weeks, clones were isolated and propagated to generate stable cell line clones.

Sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), and polyethylene glycol (PEG) were purchased from Sigma-Aldrich (Oakville, ON, Canada). Bovine serum albumin (BSA), fetal bovine serum (FBS), Dulbecco’s modified eagle medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, and penicillin/streptomycin were purchased from Wisent (St-Bruno, QC, Canada). J774 macrophage-like cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Bio-Rad Protein Assay kits were obtained from Bio-Rad Laboratories (Mississauga, ON, Canada). Lipoprint^® HDL kits were obtained from Quantimetrix (Redondo Beach, CA, USA). Extra virgin olive oil (EVOO) was obtained from Atlas Olive Oils sarl (Casablanca, Morocco).