Antibiotic antimycotic mix

A549 cells were provided by Dr. Reuben J. Shaw (Salk Institute for Biological Studies, La Jolla, CA, USA). H460, H1650, H1975, HeLa, SiHa, and HEK293T cells were purchased from ATCC (Manassas, VA, USA). H23, H1437, H2228, H2009, and H1299 cell lines were kindly provided by Dr. Massimo Broggini (Mario Negri Institute, Milan, Italy). HeLa, SiHa, and HEK293T cells were cultured in Dulbecco modified Eagle medium (DMEM; EuroClone, Milan, Italy), supplemented with 10% FCS (EuroClone), 10 mM HEPES, and 1% antibiotic–antimycotic mix (Thermo Fisher, Waltham, MA, USA). H460, H1650, H1975, H23, H1437, H2228, H2009, and H1299 cells were grown in RPMI1640 medium (EuroClone) supplemented with 10% FCS, 1% HEPES, 1% Na pyruvate, 2 mM l-Glutamine (Lonza, Basel, Switzerland), and 1% antibiotic–antimycotic mix. A549 cells were cultured in DMEM-F12 medium (Gibco-BRL, Grand Island, NY, USA) supplemented with 10% FCS, 2 mM l-Glutamine, and 1% antibiotic–antimycotic mix. In some experiments, cell lines were treated with the appropriate vehicle or with the following substances: 2 mM hydrogen peroxide (H₂O₂, Sigma-Aldrich, Saint Luis, MO, USA), 2 mM N-acetylcysteine (NAC, Sigma-Aldrich), 100 µM apocynin (Sigma-Aldrich), for the indicated time points.

The MCF-7 cells (obtained from Professor G. Leclercq, J.-C. Heuson Breast Cancer Translational Research Laboratory, Institute Jules Bordet, Free University of Brussels, Brussels, Belgium) were maintained at 37°C in a cell incubator, with a humid atmosphere of 5% CO₂. Unless specified otherwise, the cells were cultured in T-flasks, containing Dulbecco's modified Eagle's medium (DMEM), supplemented with Phenol Red, 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 25 mM N-2-hydrox yethylpiperazine-N′-2-ethanesulfonic acid, 2 mM L-glutamine and 1X antibiotic/antimycotic mix (all from Lonza, Verviers, Belgium). For the investigation of nuclear receptors by immunofluorescence microscopy, the cells were seeded in Phenol Red-free DMEM, supplemented with 10% charcoal-stripped FBS (EFM; Hyclone).

African green monkey kidney cells (VERO) and Madine Darby Canine Kidney cells (MDCK) were subcultured biweekly using Dulbecco’s modified Eagle’s medium (DMEM) (Lonza, Verviers, Belgium). The medium was supplemented with 10% Fetal bovine serum (Gibco, New York, NY, USA) and 1% antibiotic antimycotic mix (Lonza, Verviers, Belgium and incubated at 37 °C and 5% CO₂. Influenza A virus A/PR/8/34, HSV 1, Coxsackievirus B3 were obtained from Center of Scientific Excellence for influenza virus.
Influenza A virus was propagated in MDCK cell line, while HSV 1 and Coxsackievirus B3 were propagated in VERO cells. Viral titration via TCID₅₀ assay was carried out as described by Reed and Muench [58 (link)].