Ruxolitinib

Calu‐3 cells were pre‐treated with Remdesivir (Selleck Chemicals), TPCA‐1 (Bio‐Techne), PS1145 (Bio‐Techne) or Ruxolitinib (Bio‐Techne) at the indicated concentrations or DMSO control at an equivalent dilution for 1 h before SARS‐CoV‐2 infection unless otherwise stated. Inhibitors were maintained at the indicated concentrations throughout the experiments. For cytokine treatments, recombinant human IFNβ, IFNλ1, IFNλ2, IFNγ, IL1β or TNF (PeproTech) at a final concentration of 10 ng/ml was added at the indicated time points. To generate conditioned media (CoM), Calu‐3 cells were mock‐infected or infected with SARS‐CoV‐2 at 0.04 TCID50/cell and supernatants were harvested 48 hpi, clarified by centrifugation at 2,100 g for 15 min and 4°C and stored at −80°C. For conditioned media experiments, MDM were exposed to CoM as indicated, which was diluted 1:5 in RPMI, 5% FBS. After 6 h, conditioned medium was replaced with RPMI, 5% FBS and cells were harvested at 48 h for gene expression and surface marker expression analysis. MDM were treated where indicated during CoM exposure with either 2 μM Ruxolitinib (Bio‐Techne) or 2.5 μg/ml anti‐IFNAR antibody (pbl Assay Science) or an isotype control IgG2A antibody (R&D).

Human HeLa cells were treated with cholesterol-conjugated JAK1 lead compound 3033 at 1.5 μM at 37 °C for 72 h. The expression of human JAK1, JAK2, JAK3, and TYK2 were measured by the Quantigene 2.0 assay with the following probesets (ThermoFisher): human JAK1 (#SA-50455) human JAK2 (#SA-10038), human JAK3 (#SA-10158), human TYK2 (#SA-3003840), and human ACTB (#SA-10008) as a housekeeping gene. Small molecule JAK1&2 inhibitor ruxolitinib was purchased from TOCRIS (#7064, MW: 310.87 Da, C₁₇H₁₈N₆•1/4 H₂O). ruxolitinib was reconstituted according to the manufacturer’s instructions. To compare the potency of JAK1 siRNA 3033 over ruxolitinib in inhibiting the stimulation of IFN-γ signaling, HeLa cells were treated with siRNA or ruxolitinib for 72 h at a concentration of 1.5 μM followed by replacing the media containing 10 ng/mL recombinant human IFN-γ protein (R&D System, #285-IF-100) and 10 ng/mL recombinant human tumor necrosis factor (TNF)-α protein (R&D System, #210-TA-020). The expression of mRNA was measured by the Quantigene 2.0 assay with the following probesets (ThermoFisher): human JAK1 (#SA-50455) human CXCL9 (#SA-12372), human CXCL10 (#SA-50393), human CXCL11 (#SA-50464), and human ACTB (#SA-10008) as a housekeeping gene.

PVSRIPO and CBV3 infections were performed at an MOI of 10 in DMEM supplemented with 1% FBS and nonessential amino acids (39 (link), 41 (link)). Z-VAD-FMK and Ruxolitinib (Tocris) were dissolved in DMSO and used at the indicated concentrations. IFN-γ and IFN-α2 (PBL) were used at the indicated concentrations. One-step growth curves and plaque assays were performed as reported before (39 (link), 41 (link)).