Foetal calf serum

Atg5^+/+ cells and Atg5^–/– MEFs were obtained from Christian Munz, University of Zurich, Switzerland. Atg7^–/– MEFs were obtained from Stephen Tait, University of Glasgow. All cell lines in this study were cultivated in complete growth medium, comprised of Dulbecco’s modified Eagle’s medium with high glucose, pyruvate and GlutaMAX and supplemented with non-essential amino-acids, HEPES buffer, penicillin/streptomycin (all reagents, ThermoFisher Scientific) and 10% foetal calf serum (GE Healthcare, A15-502). Shield1 (Clontech, 632188) was added at a final concentration of 1 µM (stock maintained at 1 mM) in growth media. Ethanol, the solvent for Shield1, was used as a control at a 1:1000 dilution in growth media. Treatments were used at the following concentrations: chloroquine (Sigma, C6628), 50 µM; PP242 (Selleck Chemical, S2218), 1 µM; MG132 (Sigma, C2211) 10 µM; recombinant IFN-β, at the indicated concentrations (BioLegend, 581302); and thapsigargin (Sigma, T9033) 3 µg/mL. Blocking IFNAR antibody (BD Pharmingen, 561183) or isotype control (BD Pharmingen, 553447) were used at 10 µg/mL and added to culture media 1 h before infection for the duration of the experiment. Influenza A/PR/8/76 (PR8) and ΔNS1 PR8 were purchased as purified allantoic fluid or purified antigen, respectively, from Charles River Laboratories (Spafas, CT, USA).

Breast cancer cell lines BT-474 and MDA-MB-468 were purchased from ATCC. BT-474 cells were maintained at 37 °C, 5% CO₂ in Hybricare Medium (ATCC) complemented with 10% foetal calf serum (Labtech International, Ringmer, UK). MDA-MB-468 cells were maintained at 37 °C, 5% CO₂ in Dulbecco’s modified Eagle’s medium complemented with 10% foetal calf serum and 2 mM L-glutamine (PAA Laboratories, UK).

For our research, we used human fetal osteoblasts (hFOB 1.19). The cells were cultured in 175 cm2 flasks (Greiner bio-one, Germany) with DMEM-F12-medium (Invitrogen, Germany) supplemented with 10% foetal calf serum (PAA Laboratories, Germany), 105 IU penicillin and 100 mg/L streptomycin (Invitrogen, Germany) at 34 °C and 5% CO2. At a confluence of 80% the cells were harvested, washed with phosphate-buffered saline (PBS), counted and 1 × 10⁴ cells were seeded onto every specimen.

Alginate, Dulbecco’s modified Eagle medium (DMEM), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) powder, Alcian blue dye, chloramine T, 1,9-dimethylmethylene blue dye (DMB), p-dimethylaminobenzaldehyde (p-DMBA) and a DNAQF DNA Quantification Kit were purchased from Sigma–Aldrich (Poole, Dorset, United Kingdom). Foetal calf serum was purchased from PAA Laboratories (Farnborough, Hampshire, United Kingdom). All other materials were obtained from Sigma–Aldrich.

A human neonatal foreskin fibroblast cell line (HCA2 cells, provided by Prof David Kipling, University of Cardiff, UK) was cultured from frozen stock (passage 11) to 70–80% confluency (T175 flasks, Greiner Bio‐One, Frickenhausen, Germany) in Dulbecco's Modified Eagles Medium (DMEM, Invitrogen, Paisley, UK), supplemented with 10% (v/v) foetal calf serum (PAA Laboratories, Linz, Austria) and 2 mM of glutamine (Invitrogen). The supplemented medium will be referred as complete growth medium (hCGM). The cells were taken to a maximum of passage 25 over 60 days. For cell harvesting, the spent medium was removed; the cells were rinsed once with Dulbecco's Phosphate Buffered Saline (DPBS) (Sigma–Aldrich, Ayrshire, UK) and enzymatically detached using 5 mL of TrypLE™ Select (Invitrogen) for 5 min at 37°C. To quench the enzyme, an equal volume of growth media was introduced into the flask and the cells were centrifuged (Thermo, Strasbourg, France) for 3 min at 500 g and room temperature (21 ± 1°C). The cells were subsequently resuspended in ∼10 mL of growth medium to yield a suspension of ∼2 × 10⁶ cells mL⁻¹and used within 5 min for membrane separation studies.