Fetal calf serum (fcs)

All assays on specimens taken during the acute phase, harboring potentially infectious virus, were performed in the BSL-4 facility at INMI. Leukocytes were isolated by lysing red cells with NH4Cl (Sigma-Aldrich, St. Louis, MS, USA), then washed in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MS, USA) supplemented with 10% heat inactivated FCS (Euroclone, Italy) and stored at −80 °C in 90% FCS/10% DMSO (Euroclone, Italy). Cryopreserved leukocytes were rapidly thawed, washed with PBS and fixed with 10% paraformaldehyde (PFA) for 30 min at room temperature, to achieve virus inactivation. Fixed samples were transferred to BSL-2 laboratory for subsequent work.

Human lung (H157),
colon (HCT-15 and
LoVo), and pancreatic (PSN-1) carcinoma cells were obtained from the
American Type Culture Collection (ATCC, Rockville, MD, USA). Human
cervical carcinoma A431 cells were kindly provided by Prof. F. Zunino
(Division of Experimental Oncology B, Istituto Nazionale dei Tumori,
Milan). Human ovarian adenocarcinoma 2008 cells were kindly provided
by Prof. G. Marverti (Dipartimento di Scienze Biomediche, Università
di Modena University, Italy). Human colon cancer cells resistant to
doxorubicin (LoVo MDR) were kindly provided by Prof. M. P. Rigobello
(Dipartimento di Scienze Biomediche, Università di Padova,
Italy). The LoVo-OXP cells were derived, using a standard protocol,
by growing LoVo cells in increasing concentrations of OXP and following
17 months of selection of resistant clones, as previously described.^{23 (link)}Cell lines were maintained in the logarithmic
phase at 37 °C in a 5% carbon dioxide atmosphere using the following
culture media containing 10% fetal calf serum (EuroClone, Milan, Italy),
antibiotics (50 units/mL penicillin and 50 μg/mL streptomycin),
and 2 mM l-glutamine: (i) RPMI-1640 medium (EuroClone) for
A431, PSN-1, H157, HCT-15, and 2008 cells and (ii) Ham’S F-12
(Sigma Chemical Co.) for LoVo, LoVo MDR, and LoVo-OXP cells.

Human pancreatic PSN-1 and BxPC-3 carcinoma cell lines were obtained by American Type Culture Collection (ATCC, Rockville, MD, USA). Cell lines were maintained in the logarithmic phase at 37 °C in a 5% carbon dioxide atmosphere using RPMI-1640 medium (EuroClone) containing 10% fetal calf serum (EuroClone, Milan, Italy), antibiotics (50 units/mL penicillin and 50 μg/mL streptomycin) and 2 mM l-glutamine.

Cells and culture conditions: A2780 (human ovarian carcinoma) and A2780 Dox-resistant (A2780 res) cell lines were obtained from ATCC (Milan, Italy). The tumor cell lines were grown as monolayers in culture dishes in RPMI 1640 medium (Invitrogen, Burlington, ON, Canada), supplemented with 10% penicillin-streptomycin (Invitrogen) and 10% fetal calf serum (Euroclone, Milan, Italy), in a humidified atmosphere at 37 °C and 5% CO₂. To maintain resistance, A2780 Dox-resistant cell medium was supplemented with 1 μg/mL of Dox once a week.

Human lung (H157), colon (HCT-15), and breast (MCF-7) carcinoma cell lines were obtained by American Type Culture Collection (ATCC, Rockville, MD, USA). The human ovarian 2008 adenocarcinoma cells were kindly provided by Prof. G. Marverti (Dept. of Biomedical Science of Modena University, Modena, Italy). Human cervical (A431) carcinoma cells were kindly provided by Prof. F. Zunino (Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy). Cell lines were maintained using the following culture media containing 10% fetal calf serum (Euroclone, Milan, Italy), antibiotics (50 units/mL penicillin and 50 μg/mL streptomycin), and l-glutamine (2 mM): (i) RPMI for HCT-15, A431, MCF-7 and 2008 cells; (ii) DMEM medium for A375 cells.

Horse Immortal Fibroblasts [75 (link)] and HeLa (human cervical carcinoma) cells were cultured in high-glucose D-MEM supplemented with 10 % fetal calf serum (Euroclone), 2 % non-essential amino acids, 2 mM L-glutamine and 1× penicillin-streptomycin (Sigma). For primary fibroblast cell lines, the culture medium was supplemented with 20 % fetal calf serum. Cells were routinely cultured at 37 °C in 5 % CO₂.
Plasmid DNA for promoter reporter assays was prepared using QIAGEN Plasmid Midi kit. Transfections were carried out using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol.
Twenty-four hours post-transfection, cells were selected adding 300 ng/ml (horse immortal fibroblasts) or 1 μγ/ml (HeLa cells) puromycin to the medium. Cells were cultured with selective medium until the emergence of drug-resistant colonies, that is 3 weeks for horse fibroblasts and 2 weeks for HeLa cells. Pools of about 50 colonies were obtained and grown as stably transfected cell populations.