Fig. 3. Oxidation at Cys31 of FOXO3 activates IFN-I response.

IF (a), DBE reporter (b, mean ± s.e.m., n = 3 independent experiments), and WB (c) for FOXO3 in NSPCs following 24 h of respective treatment. Scale bar = 10 µm. d WB for cysteine sulfenylation (Cys-SOH) following immunoprecipitation of FOXO3. e Conserved consensus sequence adjacent to AKT phosphorylation site of mouse FOXO proteins. f WB for indicated proteins from non-treated and PQ-treated (40 μM, 0.5 h) Foxo3 WT or C31A mutant transduced NSPCs. g Microscopic analysis of Foxo3 WT or C31A mutant tagged with c-terminus EGFP with or without PQ treatment (40 μM, 16 h). Scale bar = 20 µm. Percent of cells with nuclear FOXO3-EGFP is plotted on the right. Mean ± s.e.m. of three independent experiments. qRT-PCR analysis for transcriptional targets of FOXO3 (h) and ISGs (j). Foxo null NSPCs with WT or C31A mutant Foxo3 were analyzed following 4 days of PQ treatment. Mean ± s.e.m. of three independent experiments. i IFNβ secretion in the media following 48 h treatment. Mean ± s.e.m. of three independent experiments. k Schema for activation of FOXO3 by oxidation at Cys31 residue. For b, g, h, i, and j, statistical significance was determined by one-way ANOVA. Experiments for a, c, d, f, and g were repeated three times independently with similar results and representative images/blots are shown.