Fig. 5. 4-OI impairs VSVΔ51-induced antiviral immune responses.

a–c 786-O cells pretreated with 4-OI (75 μM) for 24 h and infected with VSVΔ51 (MOI 0.01) for 17 h. RNA sequencing analysis emphasizing on antiviral genes (blue dots) in the volcano plot (a), differentially expressed interferon-stimulated genes (ISGs) in the heat map (b), and top KEGG pathways affected by 4-OI during viral infection (one-sided hypergeometric test, Benjamini–Hochberg method was applied to adjust the p-value for multiple testing) (c). d, e 786-O cells pretreated with 4-OI (125 μM) for 24 h and infected with VSVΔ51-RFP (MOI 0.01) for 24 h. IFIT1 levels assessed by fluorescence microscopy (d), and Western blot performed on cell lysates for antiviral proteins (e). f 786-O cells pretreated with 4-OI (75 μM) for 24 h infected with wild-type VSV (wt VSV) or VSVΔ51 at MOI 0.01. Viral titers determined 24 h post-infection. g–i Control and NRF2 KO 786-O cells, as well as 786-O cells transiently KO for NRF2 or KEAP using CRISPR/Cas9, pretreated with 4-OI (75 μM) for 24 h and infected with VSVΔ51 (MOI 0.01) for 17 h. Immunoblots in (g, h). CXCL10 release measured by ELISA from supernatants in (I). j Control and NRF2 KO 786-O cells pretreated with 4-OI (75 μM) for 24 h and stimulated with the RIG-I agonist M8 (3.5 ng/mL) for 5 h. Western blot performed on cell lysates. Data are from one experiment performed in triplicate in (a–c). Images are from one experiment in (d). Data are from one representative experiment performed at least three times in (e). Data are depicted as means ± SEM from one experiment performed in triplicate in (f). Data are from one representative experiment out of three in (g), out of two in (h) and (j). Data are depicted as means ± SEM from two experiments performed in triplicate in (I). Statistics indicate significance by two-way ANOVA for (f, I). Vertical stacks of bands are not derived from the same membrane in (e, g, h, j). Source data provided as a Source Data file.