Jetpei

HeLaM (Dr A. Peden, University of Sheffield, UK) and MRC-5 (ECACC) cells were cultured in DMEM supplemented with 10% FCS and 2 mM glutamine at 37 °C in a humidified, 8% CO₂ environment. Cells were transfected with DNA by addition of DNA constructs together with JetPEI (QBiogene), using half the manufacturer’s recommended amounts, followed by incubation in culture conditions for 16–24 hours. Cells were transfected with siRNA using ONTARGETplus SMARTpool siRNA duplexes (Dharmacon) together with INTERFERIN (QBiogene), according to the manufacturer’s instructions. siRNA-transfected cells were incubated for 72 hours prior to fixation, with DNA transfection carried out in fresh media after 48 hours if necessary.

The recombinant lentiviruses expressing Flag-tagged Mxi1-1 and HA-tagged Mxi1-0 were purchased from Genscript (Jiangsu, China). To prepare Mxi1-1 or Mxi1-0 overexpression viral particles, HEK293T cells were co-transfected with each viral vector and the packaging vectors (pMD2.0G and psPAX) using JetPEI purchased from Qbiogene (Montreal, Canada) following the manufacturer’s protocol. The medium was replaced 4 h after transfection, and cells were cultured for a further 36 h. Viral particles were harvested, filtered using a 0.45 µm syringe filter, and combined with 10 μg/ml polybrene (Sigma, MO, United States). PASMCs at 60% confluency were treated with these particles overnight. The culture medium was then replaced with fresh complete growth medium, and cells were cultured for a further 24 h and selected with puromycin (1 μg/ml). The selected cells were used in further experiments.

Cells from American Type Culture Collection (ATCC, Manassas, VA) were cultured at 37°C in a humidified incubator with 5% CO₂ according to ATCC protocols. Cells were cytogenetically tested and authenticated before being frozen. Each vial of frozen cells was thawed and maintained for 2 months (10 passages). Human NSCLC cells, including H460, H1650, H1299, H520, HCC827, H1975 and H358 were grown in RPMI-1640 medium supplemented with 10% FBS and antibiotics. HBE human bronchial epithelial cell was cultured with BEGM supplemented with 10% FBS and antibiotics. For transfection experiments, the jetPEI (Qbiogene, Inc., Montreal, Canada) transfection reagent was used following the manufacturer's instructions. The cells were cultured for 36–48 h and protein extracted for analysis.

Cells, including A549, H1975, HCC827, HBE, MRC5, and NL20 from American Type Culture Collection (ATCC, Manassas, VA) were cultured at 37°C in a humidified incubator with 5% CO₂. A549, H1975, and HCC827 were cultured in RPMI‐1640 medium supplemented with 1% antibiotics and 10% FBS. The MRC5 lung fibroblast cell was cultured in Eagle's Minimum Essential Medium supplemented with 1% antibiotics and 10% FBS. The HBE bronchus epithelial cell was grown in keratinocyte‐serum‐free medium with 0.05 mg/mL bovine pituitary extract, 5 ng/mL human recombinant EGF, 0.005 mg/mL insulin and supplemented with 500 ng/mL hydrocortisone. The NL20 bronchus epithelial cell was cultured in Ham's F12 medium with 2.7 g/L glucose, 1.5 g/L sodium bicarbonate, 2.0 mmol/L l‐glutamine, 0.005 mg/mL insulin, 0.1 mmol/L nonessential amino acids, 0.001 mg/mL transferrin, 10 ng/mL epidermal growth factor, 500 ng/mL hydrocortisone and supplemented with 4% FBS. The jetPEI (Qbiogene, Inc., Montreal, Canada) transfection reagent was used for transfection experiments. The cell lysate was extracted for Western blot analysis.

A673 cells and HEK293 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). SK-N-MC cells and SH-SY5Y cells were obtained from the Deutsche Sammlung für Mikroorganismen und Zellkulturen (Brunswick, Germany). 4-1BBL transgenic A673 cells and stimulation of PBMCs with anti-CD137 antibodies (clone 26G6; a kind gift from R. Mittler) was described elsewhere (10 (link)). PBMCs were isolated from healthy donors with informed consent and approval by the local ethics committee as described (45 (link)). Cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum and penicillin and streptomycin. For selection of transgenic cells, medium was supplemented with 400 μg/mL geneticin sulfate. PBMCs from HLA-A1,A2 positive donors were isolated as described (44 (link), 45 (link)). Stimulation of PBMCs and enzyme-linked immunospot (ELISPOT) analysis was performed as described (7 (link)) by using an interferon gamma ELISPOT kit (Becton-Dickinson, Heidelberg, Germany). Transfection of cells was performed using jet PEI (Qbiogene, Carlsbad, CA, USA). MACS separation was performed using anti-PE microbeads (Miltenyi, Bergisch Gladbach, Germany). Statistical analysis was performed with Microsoft Excel 2010 (Microsoft, Redmond, WA, USA).