Heat inactivated fcs

HMEC-1, carrying the type O blood group and thus not reactive with anti-blood group antibodies in human sera, were supplied by ATCC and were cultured in the MCDB 131 medium (Gibco) supplemented with 10% heat-inactivated FCS (Sigma), 2 mM l-glutamine (Gibco), 100 IU ml⁻¹ penicillin (Gibco), 100 μg ml⁻¹ streptomycin (Gibco), 10 ng ml⁻¹ epidermal growth factor (Gibco), and 1 µg ml⁻¹ hydrocortisone (Sigma-Aldrich). HUVECs were established from primary cultures as previously described⁷⁵ and cultured in Medium 199 (Gibco) supplemented with 20% FCS, 2 mM l-glutamine, 100 IU ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin, 130 μg ml⁻¹ heparin (Sigma-Aldrich), and 1.2 mg ml⁻¹ endothelial cell growth supplement (Sigma-Aldrich). CHO-K1 and xylotransferase-1-deficient CHO-K1 cells (pgsA-745 cells), which are HS and GAG deficient, were supplied by ATCC and grown in RPMI-1640 medium supplemented with 5% FCS and antibiotics. All cell lines were incubated in 5% CO₂ and ambient O₂ at 37 °C and were repeatedly tested for mycoplasma using a MycoAlert Assay Kit (Lonza).

OVCA429 parental cell line and OVISE cell line expressing tetracycline-inducible wild-type ARID1A (17 (link)) originated from the laboratory of I. M. Shih. Cell lines were tested for mycoplasma (University of Pennsylvania School of Medicine, Cell Culture Services). All cell lines were cultured on polystyrene in a 2D format in the presence of 5% CO₂ at 37 °C. OVCA429 cells were maintained in RPMI 1640 (Corning, Corning, NY, cat. no. 10–092-CM) supplemented with 10% heat-inactivated FCS (Sigma-Aldrich, St. Louis, MO, cat. no. F4135). OVISE cells expressing inducible wild-type ARID1A were maintained in RPMI 1640 supplemented with 10% Tet System Approved FCS (Clontech, Mountain View, CA, cat. no. 631107). All media contained 1% Penicillin-Streptomycin (Corning, cat. no. 30-002-CI).

1*10⁶ PBMC were stimulated with polyI:C (20 μg ml^-1, Invivogen) in 250 μl in 96-wells plates (Greiner Bio-one, Alphen aan den Rijn, Netherlands) for 5 or 7 hours at 37°C in RPMI 1640 (Invitrogen) supplemented with 9% heat-inactivated FCS (Sigma-Aldrich) and penicillin/streptomycin (Invitrogen). During the last 3 hours, cells were incubated with 10 μg ml^-1 Brefeldin A (Sigma-Aldrich). Subsequently, cells were stained for BDCA3 and CD11c, fixed with 2% formaldehyde, permeabilized with 0.5% saponin and stained for tumor necrosis factor α (TNF-α) (eBioscience), IFN-λ1 (kindly provided by Bristol-Myers Squibb and commercial Ab from R&D systems) or polyclonal goat IgG (R&D systems). Cytokine-producing cell frequencies were determined by flow cytometry.
Isolated DCs were stimulated for 24 hours with 20 μg ml^-1 polyI:C in the presence of 10 ng ml^-1 GM-CSF. Levels of secreted human IFN-λ1 (interleukin 29; IL-29) were measured using a commercially available ELISA kit (eBioscience) and IL-1β, IL-6, IL-8 and TNFα levels were measured using a BD cytometric bead array (CBA, BD Biosciences). Detection limits were 8 pg ml^-1 (IFN-λ1), 7.2 pg ml^-1 (IL-1β), 2.5 pg ml^-1 (IL-6), 3.6 pg ml^-1 (IL-8), 3.7 pg ml^-1 (TNF-α).

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood from H. pylori negative healthy donors, after informed consent, by density gradient centrifugation with Biocoll (Biochrom, Germany). Monocytes were isolated from PBMCs by magnetic cell labeling (MACS) with the Monocyte Isolation Kit II (Miltenyi Biotec, Germany) following the manufacturer's instructions. Their purity was determined by flow cytometry staining (anti-CD14 and anti-CD45).
Immature DCs were generated by culturing monocytes in RPMI 1640 with Glutamine (Invitrogen, USA), 10% heat-inactivated FCS (Sigma, USA) and 1% Penicillin/Streptomycin, (Invitrogen, USA), 20 ng/ml human rIL-4 (Miltenyi Biotec, Germany) and 20 ng/ml human rGM-CSF (Miltenyi Biotec, Germany) for 6 days.

The human A549 (ATCC^® CCL-185™) lung epithelial adenocarcinoma cell line was purchased and authenticated from the ATCC bank (Manassas, VA, USA). For the experiments, the cells were seeded in 5 × 10⁵ in 6-well plates (Greiner Bio-one, Kremsmünster, Austria) with 2 mL medium (See Table S2) containing 100 mg/dL glucose (1:1 mix of Gibco™ RPMI 1640 mediums (Thermo Fisher Scientific, Waltham, MA, USA) containing either 0 mg/dL or 200 mg/dL glucose, supplemented with 10% heat-inactivated FCS (Sigma-Aldrich, St. Louis, MO, USA), 1% Penicillin–Streptomycin (Anprotec, Bruckberg, Germany), or 1% 2 mM L-Glutamine (Anprotec, Bruckberg, Germany). The cells were infected with RV-A1b as described below and/or stimulated with poly I:C (working concentration: 20 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) at different time points relative to the RV-A1b infection (see experimental design). Cells were incubated at 37 °C, 5% CO₂ and 96% humidity. They were cultured for 48 h for RNA analysis and supernatant, and 72 h for supernatant and flow cytometry analysis. Cells were detached using Trypsin-EDTA (Anprotec, Bruckberg, Germany) and then counted using Trypan Blue solution (Sigma-Aldrich, St. Louis, MO, USA) in a Neubauer chamber in accordance with the formula ((Q1 + Q2)/2) × 2 × 10,000 × x mL.