Hepes buffer

Huh7.5 hepatoma cells (Apath LLC) (54 (link)) were maintained in DMEM with 10% FBS, 100U/ml Penicillin/Streptomycin, 1X non-essential amino acids, 1X sodium pyruvate and 1X Glutamax (all Gibco/Life Technologies). For virus production and GLuc assays, medium was supplemented with 1 μM vitamin E (α-tocopherol) (Sigma) and 50 mM HEPES buffer, pH7.4 (Cellgro) (19 (link)). The cell culture infectious subtype 1a HCV genome H77S.3, and its derivative H77S.3/GLuc2A, encoding a GLuc reporter gene, were both described in (10 (link)). The cell culture infectious subtype 1b HCV genome N.2, and its derivative N.2/GLuc2A, encoding a GLuc reporter gene, were both described in (55 (link)). The Q80K amino acid substitution was generated in H77S.3 and N.2 backgrounds using the Quikchange Site-Directed Mutagenesis Kit (Agilent).

For creation of a random mutant NS1 reporter cell library (HeLa-NFκB-DsRed2-pLEX-mNS1), HeLa-NFκB-DsRed2 cells were transduced at an MOI of 0.02 with pLEX-mNS1 particles to ensure fewer than 1 integration event per cell. HeLa-NFκB-DsRed2 cells were plated in 6-well plates and overlaid with 2ml of 1+1+1 DMEM (1X DMEM (CellGro) with 1% Pen/Strep (CellGro), 1% HEPES buffer (CellGro), 1% FBS (Gemini)), containing 8μg/ml polybrene (Sigma), and lentivirus-containing supernatant. Medium was changed 24h post-transduction to 1X DMEM with 10% FBS and cells were placed under puromycin selection (2μg/ml) (Gemini) 48h later. HeLa-NFκB-DsRed2 cells or standard HeLa cells were also transduced with either pLEX-NS1 or pLEX-MCS lentivirus particles to serve as controls.
Lentivirus titers were determined by counting crystal violet-stained, puromycin-resistant HeLa cell colonies following transduction with serial dilutions of lentivirus-containing supernatants.

Human peripheral blood mononuclear cells (PBMCs) from healthy volunteers (20–60 years of age), patients with CD (25–60 years of age), and patients with CD taking 6-MP (25–58 years of age) were isolated using SepMate™ and Lymphoprep™ (Stemcell Technologies, Vancouver, BC, Canada) density gradient medium according to the manufacturer’s protocol. NK cells were purified from PBMCs by negative selection technique using EasySep™ (Stemcell Technologies) human NK cell enrichment kit according to the manufacturer’s protocol. The purity of isolated NK and T cells were confirmed by flow cytometry to be above 90%.
Cultures of NK cells were performed in complete medium composed of RPMI-1640, 10% heat inactivated FBS, 100 IU mL⁻¹ penicillin and 100 μg mL⁻¹ streptomycin, 2 mM L-glutamine, 10 mM HEPES buffer (Cellgro, Manassas, VA), 5 × 10⁻⁵ M 2-ME (Sigma-Aldrich, St. Louis, MO), and 1 ng mL⁻¹ (13 IU) recombinant human IL-2 (R&D Systems, Minneapolis, MN) [31 (link)]. NK cells were cultured at 5–10 × 10⁴ cells mL⁻¹ in 96-well plates for 24–72 h. For cell survival and Rac-1 assays, cells were cultured with 5–25 μM of 6-MP (Sigma Aldrich), 5–10 μM of 6-TG (Sigma Aldrich), or 100 μM of Rac1 inhibitor ITX-3 (Sigma Aldrich) to mimic physiologic levels of 6-MP and 6-TG as previously described [26 (link)].

Murine bone marrow macrophages (BMM) were differentiated from the bone marrow of six to eight week old female or male C57BL/6J mice and maintained in culture with bone marrow macrophage medium (BMM medium - DMEM (CellGro) + 1 mM Sodium Pyruvate (CellGro) + 1 mM HEPES Buffer (CellGro) + 2 mM L-glutamine (CellGro) + 20% heat inactivated FBS (Seradigm) + 30% L929 fibroblast cell supernatant + 100 μg/ml Penicillin/Streptomycin (Pen/Strep) (CellGro) + 50 μM β-mercaptoethanol (Amresco) at 37°C, 5% CO_2. Murine bone marrow neutrophils were isolated from the bone marrow of six to eight week old female or male C57BL/6J mice and maintained in culture with DMEM (Corning) + 10% heat inactivated FBS supplemented with 100 μg/ml Penicillin/Streptomycin at 37°C, 5% CO_2. NFκ^B reporter macrophages (RAW-Blue – Invivogen) were maintained in culture with DMEM (Corning) + 10% heat inactivated FBS supplemented with 100 μg/ml Penicillin/Streptomycin at 37°C, 5% CO_2.