Fig. 2.

Scratch wound assay defines candidates for oxidation-dependent repair. (A) Effects of inhibitors on HEK001 scratch closure (5 µM sets shown). Inh., inhibitor. (B) Dose response of inhibitors measured using the scratch closure distance between the two edges of the wound. (C) HPF signals at the scratch margin in the absence and presence of FOXO1 inhibitor. SC, scratch. Time after scratching is shown in the top right. 30ʹ, 30 minutes. (D) Rapid nuclear localization of FOXO1 at the scratch margin of vehicle controls. Pre-treatment with DPI decreases FOXO1 staining at the scratch. Inset shows magnification of cells marked with red arrowheads. (E) Nuclear NF-κB1 (p50 and p105; p50/p105) is noticeable at the wound margin at 6 h post scratching but is cytoplasmic following wound closure at ∼18 h, similar to in unscratched (unscratch) cells. (F) Predominantly cytoplasmic NF-κB1 p50/p105 staining was found in untreated cells and following H₂O₂ treatment for 2 h. (G) Non-canonical NF-κB2 (p52 and p100; p52/p100) immunofluorescence following scratching shows predominantly cytoplasmic staining. (H) p52/p100 immunofluorescence reveals sustained cytoplasmic localization after H₂O₂ treatment for 2 h. High H₂O₂ concentrations lead to increased expression of NF-κB2. Scale bars: 100 µm (A-E,H); 50 µm (F,G). Two-way ANOVA and Bonferroni's multiple comparison post hoc tests were used. Significance: *P<0.05, ***P<0.001, ns: not significant (n≥3-5 cell culture experiments).