Fig. 2: CAD triggers a pro-inflammatory DNA-damage response.

A HaCaT cells (control or ICAD-mAID-GFP) were treated with solvent or auxin (20 µM) for 24 h. Cells lysates were analyzed by Western Blot. Blots are representative of 3–5 experiments. B HaCaT ICAD-mAID-GFP cells were treated with solvent or auxin for 6 h. Auxin was washed out and culture was continued for 24 h. As a positive control for p65-phosphorylation, cells were treated with TNF (100 ng). Cells were harvested and analyzed by Western Blot for p-p65 and p100/p52 (representative of three separate experiments). C HeLa cells (control or ICAD-mAID-GFP cells) were treated with solvent (DMSO (-)) or auxin (5 µM) for 6 h. Auxin was washed out and culture was continued for 24 h. Cell lysates were analyzed by Western blotting for NF-κB p-p65 and p100/p52. A blot representative of three independent experiments is shown. D RT-qPCR analyses of TNF and IL-6 mRNA levels in HaCaT ICAD-mAID-GFP cells after auxin (20 µM) treatment. Data represents means/SEM of at least four independent experiments (each symbol shows one experiment). Data were normalized to GAPDH and then to the DMSO control (-). Statistical significance was tested by two-way ANOVA, Tukey’s post-hoc test. E HaCaT cells were treated with solvent or auxin (20 µM) for 6 h. Auxin was washed out and culture was continued for 48 h. Supernatants were collected and IL-6, IL-8 and CXCL-1 were measured by ELISA. Shown are means/SEM of at least three experiments. Significance was determined by two-way ANOVA, Sidak’s post-hoc test. F, G HeLa control cells or HeLa ICAD-mAID-GFP cells were treated with auxin (5 µM) or DMSO for 6 hours, then auxin was washed out and culture was continued for 48 hours. Cell culture supernatants (1:2 (donor 1 or 1:5 (donor 2) dilutions) were used in transwell assays with human neutrophils isolated from two healthy donors to measure chemotaxis (F) or neutrophil survival by AnnexinV/PI staining (G). Data are means/SEM of three independently derived supernatants. Significance was tested by two-way ANOVA, Sidak’s multiple comparisons test. For all samples: ns, p ≥ 0.05, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.