Fig. 1: CAD contributes to cytokine induction through sub-lethal signals, and activation of CAD causes transient, non-lethal DNA-damage and DDR.

A HeLa cells (control or deficient in CAD) were treated with the BCL-2/BCL-X_L/BCL-w-inhibitor ABT-737 (10 µM) or the Mcl-1 inhibitor S63845 (100 nM) for 72 h. IL-6, IL-8 and CXCL-1 concentrations were determined in the supernatants. Data means/SEM of at least three experiments. Significance was determined by two-way ANOVA, Sidak’s post-hoc test. B Western blots showing induction of DDR-phosphorylation events after 6 h auxin (20 µM) treatment of HaCaT ICAD-mAID-GFP cells or control cells (Ctrl). The blot shows one of three very similar results. C Quantification of γH2AX-positive cells after 6 h auxin-treatment (20 µM) as detected by immunofluorescence microscopy. Three separate experiments were performed and analyzed. For each sample, 5 random pictures were taken and a minimum of 80 cells were quantified. Graph shows the mean value/SEM of the three experiments. Statistical analysis was done with a paired two-sample t-test. D HaCaT cells were treated with solvent (DMSO) or auxin (20 µM) for 24 h. Cells were stained with propidium iodide (PI) and analyzed by flow cytometry. Shown are means/SEM of at least 4 experiments. Significance was tested by two-way ANOVA, Sidak’s post-hoc test. ns, p ≥ 0.05. E Alkaline comet assay to assess DNA-damage. HaCaTICAD-mAID-GFP cells were treated for 6 h with auxin (20 µM) and analyzed either directly or after 24 or 48 h following auxin wash-out. Each symbol represents one cell. Data are from three separate experiments, and at least 70 nuclei were assessed per condition. Statistical analysis was done using nested one-way ANOVA with Sidak’s multiple comparisons test. Tail moment = tail DNA [%] x tail length. F Time course of γH2AX-positivity after CAD activation in HaCaT (Control) and HaCaT ICAD-mAID-GFP cells under the conditions described in (E). Signals were quantified from Western blots and are shown as means/SEM of n = 3 independent experiments. Significance was determined by two-way ANOVA, Sidak’s post-hoc test. For all samples: ns, p ≥ 0.05, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.