Fig 2: IEG induction is independent of DSBs or stress(A) TOP2A and TOP2B covalently bound to genomic DNA (ICE) under 30 min of vehicle (DMSO), Merb (200 µM), serum (1%), or Etop (20 µM) treatments. A representative image is shown (n = 3).(B) Quantification of ?H2AX foci immunofluorescence upon 30 min of the indicated conditions (40 cells from each experimental condition were manually counted, double-blind) (n = 3). Individual experimental values and mean ± SEM are shown. One-way ANOVA test with Dunnett’s post-test.(C) ?H2AX ChIP at the indicated regions of c-FOS gene in serum-starved RPE-1 cells, as measured by qPCR, at 30 min following vehicle (DMSO), Merb (200 µM), serum (1%), or Etop (50 µM) treatments (n = 3). Individual experimental values and mean ± SEM are shown. Two-way ANOVA test with Dunnett’s post-test.(D) c-FOS mRNA levels upon 30 min of the indicated conditions (n = 3); details as in Figure 1C. One-way ANOVA test with Dunnett’s post-test.(E) Quantification of phosphorylated H3S10 (pH3S10) immunofluorescence in serum-starved RPE-1 cells treated for 30 min with vehicle (DMSO), Merb (200 µM), or serum (1%) (n = 3). Individual cellular values and median are shown. Kruskal-Wallis test with Dunn’s post-test.(F) Representative image (top) and quantification (bottom) of p-p38 MAPK (Thr180/Thr182) western blot upon 30-min incubation with vehicle (DMSO), Merb (200 µM), and H2O2 (100 µM or 1 mM) (n = 3). Individual experimental values and mean ± SEM are shown. One-way ANOVA test with Dunnett’s post-test.(G) c-FOS mRNA levels upon 30 min of vehicle (DMSO), Merb (200 µM), or H2O2 (100 µM) (n = 3); details as in Figure 1C. One-way ANOVA test with Dunnett’s post-test.In all cases, only the p values indicating statistically significant differences against vehicle treatment are shown.