IFNα induces apoptosis in ISG15 KO cells. (A) Healing/migration ability of WT and ISG15 KO fibroblasts was assessed with the scratch assay. Left; percent confluency was recorded every hour for a total of 93 h. Right; experimental replicates at 48, 72, and 93 h after scratch. (B) Representative images of WT and ISG15 KO fibroblasts at 1 h (left), 49 h (middle), and 92 h (right) of 1,000 IU ml⁻¹ IFNα treatment. White arrows indicate macroscopic evidence of cellular stress. (C) WT and ISG15 KO fibroblasts were treated with IFNα2b (0, 100, 1,000 IU/ml) for 72 h, and cell death was quantified by measuring Annexin V and Propidium Iodide (PI) levels through flow cytometry. (D) Control and ISG15 KO fibroblasts were treated with IFNα2b (0, 100, 1,000 IU/ml) for 72 h following a 30-min pretreatment with necrostatin-1, and cell death was quantified by measuring Annexin V and PI levels through flow cytometry. (E) Metabolic activity of two control and one ISG15 KO fibroblast cell lines was assessed with the Deep Blue assay. Nec-1; necrostatin-1. (F) Control and ISG15-deficient fibroblasts were treated with either IFNα2b (1, 100, 1,000 IU/ml) or chloroquine (0, 25, 50, 100 μM) for 72 h. Cell lysates were analyzed by western blotting for autophagy (LC3B). (G) Control and ISG15-deficient fibroblasts were treated with either IFNα2b (100 IU/ml), or TNFα (5 ng/μl), or both for 72 h, and percent death was assessed with the Deep Blue assay as compared to the nontreated condition for each variant. (H) WT and ISG15 KO fibroblasts were treated with IFNα2b (0, 100, 1,000 IU/ml) for 72 h, and cell death was quantified by measuring cleaved (active) caspase 3 levels through flow cytometry. Source data are available for this figure: SourceData F7. P values were calculated with two-tailed t test. **P < 0.01; ***P < 0.001.