Recombinant ifn β

RPMI-1640 and IMDM media were supplemented with 10% v/v heat-inactivated FCS (Atlanta Biologicals, Flowery Branch, GA, USA), 0.1 mM non-essential amino acids, 2 mM l-glutamine, sodium pyruvate, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME (Gibco). Recombinant IFN-β and IFN-α were purchased from PBL Assay Science. Blocking anti-IFN-AR1 mAb was from Leinco Technologies (St. Louis, MO, USA). Recombinant IL-10 and IL-6 were from PeproTech (Rocky Hill, NJ, USA). Jak inhibitors Tofacitinib and Ruxolitinib were purchased from LC Laboratories (Woburn, MA, USA).

CSCs were treated with 500 U/mL of recombinant IFN-β (PBL Assay Science, Piscataway, NJ, USA). Pictures were taken on experimental days 0, 2, and 7. Sphere size was measured with CellProfiler 3.1.9 (Broad Institute, Cambridge, MA, USA), objects higher than 1500 pixels were used for the analysis. Total RNA was isolated at 24 h for further gene expression analysis.

Cells were infected with wt J2315 at an MOI of 1 in a 96-well plate, with 25,000 cells/well in 100μL antibiotic-free complete DMEM. At the appropriate time, media was removed from infected cells and wells were washed 3x with warm, sterile PBS. Cells were then lysed in 100 μL of H₂O for 10 min at RT. Lysates were then serially diluted 1:10 in sterile PBS down to a 10⁻⁶ dilution. These dilutions were then plated on tryptic soy agar plates supplemented with 5% sheep’s blood and allowed to grow overnight at 37°C. Colonies were counted and counts were converted to their original concentrations from the cell lysate. For IFN pre-stimulation assays, 1000 U/mL recombinant IFN-β (PBL Assay Science) or 100 ng/mL recombinant IFN-γ (PBL Assay Science) was added for the given times before the removal of IFN-containing media and replacement with media containing J2315. Infections then proceeded as explained above.

HEK-293T were plated in 24-well plates (5 × 10⁵ cells per well). One day later, HEK-293T cells were co-transfected (JetPRIME; Polyplus) with IFN-β-pGL3 or pISRE-Luc plasmid (0.3 μg/well; Stratagene) that contain the firefly luciferase reporter gene downstream of an IFN-β-specific promoter sequence or the ISRE enhancer element upstream, respectively. Cells were simultaneously co-transfected with the pRL-CMV reference plasmid (0.03 μg/well; Promega) and the empty pCI-neo-3×FLAG expression vector (0.3 μg/well) or encoding proteins as specified. When specified, cells were transfected with 0.1 μg/well of poly(dA:dT) (Invivogen) or treated with 1 x 10³ IU/ml of recombinant IFN-β (PBL Assay Science) 24h after transfection. After 24h post-IFN-β-stimulation or 48h post-transfection, the cells were lysed (Passive lysis buffer, Promega), and both firefly and Renilla luciferase activities in the lysate were determined using the Bright-Glo and Renilla-Glo luciferase assay systems (Promega), respectively. The reporter activity was calculated as the ratio of firefly luciferase activity to the reference Renilla luciferase activity. All graphs show mean values and include error bars indicating the standard deviations (SD).