Mccoy s 5a medium

For all experiments colon carcinoma cell line HCT116 (ATCC, CCL-247) was used. Cells were cultured in McCoy’s 5A medium (Lonza, Basel, Switzerland) supplemented with 10% FBS and antibiotics. For normoxic conditions cells were incubated with 21% O₂ and 5% CO₂. For hypoxic conditions cells were kept in hypoxic incubator (Toepffer Lap Systems, Göppingen, Germany) with 1% O₂ and 5% CO₂. For cell cycle synchronization in G2/M phase cells were treated with 100 ng/ml nocodazole 16 h before lysis. Controls were treated with DMSO as vehicle. Treatment with 1 mM insulin took place 3 h before lysis. All experiments were performed with mycoplasma-free cells.

The following breast cancer cell lines were used in this study: SK-BR-3, MDA-MB-453, MDA-MB-231 and T-47-D (American Type Culture Collection, Manassas, VA, USA). The MDA-MB-453, MDA-MB-231 and T-47-D cells were cultured in Dulbecco's Modified Eagle Medium, DMEM (Sigma-Aldrich, Saint Louis, MO, USA), and the SK-BR-3 in the McCoy’s 5A medium (Lonza, Basel, Switzerland). All media contained 2 mM l-glutamine and were supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA) and 1 x penicillin-streptomycin solution (Biowest, Nuaillé, France). The cells were cultured at 37 °C in 5% CO₂ and humidified atmosphere. Upon reaching 90% confluence, the cells were passaged using Trypsin-EDTA solution (Thermo Fisher Scientific, Waltham, MA, USA)

Human colon cancer cell line H116 and human ovarian cancer cell line SKOV3 were obtained from the American Type Culture Collection (ATCC). H116 cells were cultured in Dulbecco's Modified Eagle's medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Scientific, Waltham, MA), 10 units/ml penicillin, and 10 mg/ml streptomycin, and SKOV3 cells were cultured in McCoy's 5A medium (Lonza, Walkersville, MD) supplemented with the same. Immortalized MEF lines were generated as previously described [29 (link)]. Cells were cultured at 37°C in a humidified atmosphere of 5% CO₂.

HER2+ breast cancer cell lines BT474 (HTB-20) representing Luminal B (ER+; PR+, HER2+; ductal carcinoma) subtype and SKBR3 (HTB-30) representing HER2+ subtype (ER-; PR-; HER2+; adenocarcinoma) of breast cancer were purchased from the American Type Culture Collection (ATCC) [25 (link),26 (link)], which were known to well characterized in previous studies and decribed highly sensitive to trastuzumab treatment [27 (link),28 ]. SKBR3 cells were maintained in Mc Coy’s 5A medium (Lonza) with L-glutamine containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin. BT474 cells were maintained in RPMI 1640 medium with L-glutamine (Lonza) supplemented with 10% FBS, 1% penicillin-streptomycin and 2% bovine insulin. The cell lines were cultured in a humidified air supplemented with 5% CO₂ at 37°C. Trastuzumab was (kindly gifted by Prof. Dr. Hakan Gürdal from the Department of Pharmacology in Medical School of Ankara University) dissolved in phosphate-buffered saline (PBS) (stock concentration of 300 mg/mL) and stored at 4°C.

For assessment of NK cell function, donor control PBMCs or CBMCs were plated in RPMI (Lonza, Slough, UK) containing 10% heat-inactivated fetal calf serum (FCS) supplemented with 1% penicillin and streptomycin (complete media) and containing human IL-2. Test cultures using 50% CBP diluted with complete media or complete media only contained 200 IU IL-2/well in 96-well plates. All cultures were incubated at 37°C with 5% CO₂ for 48 h before PMA and ionomycin stimulation. Duplicate cultures were carried out for each experiment without PMA and ionomycin stimulation to assess levels of NKG2D and baseline CD107a. NKG2D expression on the relevant cells was determined using the unstimulated cultures and CD107a background levels in unstimulated cultures were subtracted from the values obtained for equivalent stimulated cells.
Experiments were carried out using four different, healthy PBMC donors, each tested with CBP samples from the 181 UCB units and data points represent donor means. For experiments using CBMCs, four CB units were used with seven different CBP samples. HCT116 (human colon carcinoma) used for luciferase assays were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK). HCT116 cells were cultured in McCoy’s 5A medium (Lonza, Slough, UK) supplemented with 2 mM l-glutamine and 10% FCS at 37°C with 5% CO₂.

The breast epithelial cell lines ME16C2 [55] (link), and SUM102, a gift from Steve Ethier of Wayne State University (Asterand, MI, USA), and their derivatives were grown in TCF-EGM developed by the Tissue Culture Facility at the University of North Carolina at Chapel Hill (http://www.unclineberger.org/tcf/index.asp). The luminal breast cancer cell lines ZR-75-1 (CRL 1500; ATTC, Manassas, VA, USA) and MCF-7 (HTB-22; ATTC) and their derivatives were grown in RPMI1640 with phenol red (Lonza, Walkersville, MD, USA) supplemented with 2 mM L-glutamine (Invitrogen, Carlsbad, CA, USA) and 10% FBS (Invitrogen). HCT116 (CCL-247; ATTC) was grown in McCoy’s 5a medium (Lonza) supplemented with 10% FBS. For the hypoxia experiments cells were grown in a hypoxic chamber, MIC-101, (Billups-Rothenberg, Del Mar, CA, USA) at 1% O₂.
Transductions were performed as previously described [27] (link). Stably transduced cell populations were established by selection with puromycin (2 µg/mL; Invitrogen) for 2–5 weeks. All cell populations were analyzed by Western blotting for expression of NDRG1. Cell populations transduced with the vector pSiRPG [27] (link) without insert were also created as controls.