Fig. 4. 4-OI enhances VSVΔ51 infectivity independently of NRF2 and KEAP1 in cancer cells.

a NRF2 protein levels in control and NRF2 knockout (KO) 786-O cells treated with 4-OI (75 μM) for 24 h using confocal microscopy. Blue: actin filaments, green: NRF2. Scale bars, 50 μm. b KEAP1 levels analyzed in 786-O cells treated with alkynated 4-OI (4-OI-alk) (125 μM) for 4 or 24 h with or without non-alkynated 4-OI (125 μM) by anti-KEAP1 immunoblotting. c Viral RNA content assessed by RNA sequencing in VSVΔ51-infected (MOI of 0.01) control and NRF2 KO 786-O cells with or without 4-OI (75 μM) pretreatment. d–f Immunoblot analysis in control and NRF2 KO cells pretreated with 4-OI (75 μM) before VSVΔ51 challenge (MOI of 0.01) (d). Fluorescence microscopy showing VSVΔ51-RFP spread and cellular layer integrity with Hoechst stain overlay (Scale bars, 300 μm) (e) and quantification of infected cells by flow cytometry at 17 h post-infection (f). g–j Quantification of virus-infected cells by flow cytometry in 786-O cells transiently KO for NRF2 (g) or KEAP1 (j). Immunoblot analysis in NRF2 KO (h) and KEAP1 KO cells (i) pretreated with 4-OI (75 μM) before VSVΔ51 challenge (MOI of 0.01). k 786-O cells incubated with L-NAC (1 mM) for 3 h before 4-OI challenge (75 μM) for 24 h, then infected with VSVΔ51-RFP (MOI of 0.01). Quantification of infected cells by flow cytometry at 17 h post-infection. Data are means ± SEM from two independent experiments in biological duplicates and triplicates in (f), and in biological triplicates and quadruplicates in (g). Two experiments in biological quadruplicates in (j) and (k). Images from one representative experiment in triplicates in (a) and (e). Data in (b) and (d) from one representative of three independent experiments. Data as means ± SEM from one experiment in biological triplicates in (c). Statistics indicate significance by one-way ANOVA for (f, g, j, k). Vertical stacks of bands are not derived from the same membrane in (d, h, I). Source data provided in the Source Data file.