Recombinant IFNλ4 induces ISGs but not cell death. (A) Schematic of the plasmid of the inducible expression of rhIFNλ4p179 (rhIFNλ4) in a Drosophila S2 Schneider cell expression system. (B) Immunoblot of rhIFNλ4 in lysates, neat supernatants, and supernatants concentrated by TCA treatment upon treatment with or without copper (II) sulfate (CuSO₄) for 8 d. (C) Coomassie stain and immunoblot of rhIFNλ4 purified from S2 cell supernatant by affinity chromatography and gel filtration. Lanes: M, molecular weight marker; 1, purified rhIFNλ4; 2, prepurified supernatant. (D) EC₅₀ curve of rhIFNλ3 and rhIFNλ4 activity assayed by MX1 gene expression. (E) Immunoblot of pSTAT1 in wild-type and IFNLR1^−/− PH5CH8 cells, treated with rhFNβ, rhIFNλ3, and rhIFNλ4. Total STAT1 and β-actin are shown as loading controls; total STAT1 was probed on a separate immunoblot from the same lysates as pSTAT1 and STAT1 antibodies were raised against the same species. (F–H) Gene expression of MX1 (F), OAS1 (G), and ISG15 (H) in wild-type and IFNLR1^−/− PH5CH8 cells treated with recombinant rhIFNβ, rhIFNλ3, or rhIFNλ4 for 6 h. (I) Proliferation and cell death (object counts) in wild-type PH5CH8 hepatocytes stimulated with rhIFNλ3, rhIFNλ4, and ActD (positive control), measured using Incucyte time-lapse live fluorescent microscopy of a quantifiable cell viability dye, Sytox green, which marks dead cells. (J) MX1 gene expression measured at 70 h to confirm stimulation of the hepatocytes by rhIFNλ3 and rhIFNλ4 in cell death assays. Experiments are representative of at least two to three biological replicates.