Hela cells

SM from Silybum marianum seeds was prepared according to Kahol et al. [49 ]. The SM did not contain any solvent residue. The same bioactive flavonolignans were determined as in the standards with the help of a HPLC-MS method [4 (link)]. P60 was obtained from Sigma-Aldrich Buchs (St. Gallen, Switzerland). TC was a kind gift from Gattefossé (Lyon, France). Cremophor A6, A25 was obtained from BASF (Ludwigshafen, Germany). Sucrose esters (SP 50, SP 70, PS 750) were kind gifts from Sisterna (Roosendaalc, The Netherlands). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s Modified Eagle’s Medium (DMEM), Hank’s Balanced Salt Solution (HBSS), phosphate buffered saline (PBS), Trypsin-EDTA, Heat-inactivated fetal bovine serum (FBS), l-glutamine, non-essential amino acids solution, penicillin-streptomycin, were purchased from Sigma-Aldrich). 96-Well cell plates, 12 well cell plates and culturing flasks were obtained from Corning (Corning, New York, NY, USA). Cetostearyl alcohol, stearic acid, propylene glycol, IPM, nipagin M were supplied by Hungaropharma Ltd., (Budapest, Hungary). HeLa cells (human cervical cancer cells) were obtained from the European Collection of Cell Cultures (ECACC, Public Health England, Salisbury, UK). HaCaT cells (human keratinocyte cells) were obtained from Cell Lines Service (CLS, Heidelberg, Germany).

HeLa cells were purchased from the European Collection of Cell Cultures (UK) and U2OS cells were a gift from Dr Gyula Timinszky (LMU, Munchen, Germany). HeLa and U2OS cells were maintained at 37 °C under 5% CO₂ in Dulbecco’s Modified Eagle Medium (DMEM, #E15-009, PAA) supplemented with 10% fetal bovine serum (FBS, #A15-101, PAA), 1% penicillin/streptomycin (P/S, #15140-122, Gibco, Life Technologies) and 1% L-Glutamine (Glu, #25030024, Gibco, Life Technologies). For live cell imaging, cells were seeded on Lab-Tek 4 wells (#055078, Dominique Dutscher) at 60% confluence 24 h before transfection on day 1. On day 2, transfections were carried out using JetPrime reagent (#114-15, Polyplus) according to the manufacturer’s instructions. After 4 h of incubation with the transfection mixture, medium was replaced by 400 μl per well of culture medium composed of FluoroBrite phenol red-free medium (#A18967-01, Life Technologies) containing 0.1% FBS for overnight starvation. For experiment relating to Fig. 2, U2OS cells were transfected with 0.5 μg/well of LSSmOragne-mKate2 plasmid, and 0.25, 0.5, 0.75, and 1 μg/well of the different mTFP1-Yellow plasmids, in order to generate cells with an array of tandem constructs expression levels.

HeLa cells obtained from the European Collection of Cell Cultures (ECACC; Sigma-Aldrich) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with L-glutamine and glucose, 10% fetal calf serum, in a humidified incubator (LEEC Ltd, UK) at 37 °C and 5% CO₂.

Human epithelial carcinoma (HeLa) cells were obtained from the European Collection of Cell Cultures (Salisbury, UK) and maintained in DMEM supplemented with GlutaMAX I and 10% fetal bovine serum (FBS, Invitrogen), 10% charcoal stripped FBS (CSS, Gibco), or lipoprotein deficient FBS (LDS, Sigma) in a humidified atmosphere of 5% CO₂ at 37 °C.

Human cervix carcinoma HeLa cells were obtained from the European Collection of Cell Cultures (ECACC). The cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, Budapest, Hungary), supplemented with 10% fetal bovine serum (Biowest, Nuaillé France), 4 mM L-glutamine (Sigma-Aldrich, Hungary), 100 U/mL penicillin, 100 µg/mL streptomycin solution (Sigma-Aldrich, Budapest, Hungary) and 0.25 µg/mL amphotericin B (Sigma-Aldrich, Budapest , Hungary) in a humidified atmosphere containing 5% CO₂ at 37 °C. On reaching 80% confluence, cells were detached every 2–4 days using 0.05% (w/v) trypsin, 0.02% (w/v) EDTA solution and not used beyond passage 20.

Supernatants from RV16-infected HBECs were collected 24 h post infection and 1:2 serial diluted in DMEM with Glutamax containing 2% FBS, 1% penicillin-streptomycin, 1% non-essential amino acids and 1% sodium pyruvate (Life Technologies, Carlsbad, CA, USA). Dilutions were added to 80% confluent Ohio HeLa cells (European Collection of Cell Cultures) in 96-well plates (Nunc Technologies, Carlsbad, CA, USA) in duplicates. Plates were rocked for 1 h at room temperature and then incubated for 4 days at 37 °C, 5% CO₂. Then the cell monolayer was fixed and stained with crystal violet. Cytopathic effects were assessed by spectrophotometry and TCID₅₀ was calculated with the Spearman-Kärber algorithm.