Fig 2: The ZBTB38 protein accumulates in H2O2-treated cells, and the accumulation depends on ROS and USP9X activity. (A) Western blot analysis in HeLa cells exposed to H2O2 at the indicated concentrations for 30 min. P-H2AX: phosphorylated H2AX. Hydroxyurea treatment (HU) was used as a positive condition for H2AX phosphorylation. (B) ZBTB38 mRNA levels in samples from panel A. qRT-PCR normalized with three housekeeping genes (MAPK14, TBP and TFRC), n = 3. (C) Left panel: treatment of HeLa cells with Etoposide (Etop.; 200 µM for 1 h) or Hydroxyurea (HU; 2 mM for 2 h) causes DNA damage, but no ZBTB38 accumulation. Right panel: Etoposide or Hydroxyurea do not increase the intracellular ROS levels, as measured by DHE staining. The mean DHE intensity is indicated on the graph. (D) Extracellular Superoxide Dismutase (SOD) or Catalase (Cat.) do not prevent ZBTB38 accumulation in response to H2O2 treatment (200 µM for 30 min), but PEG-coupled SOD or Cat., which are cell-permeable, do prevent ZBTB38 accumulation. (E) USP9X, but not USP7, accumulates in cells treated with H2O2 (200 µM for 30 min). (F) ZBTB38 protein does not accumulate in response to H2O2 in cells depleted of UPS9x. (G) Inhibition of USP9X by WP1130 prevents ZBTB38 accumulation in response to H2O2 treatment.

Fig 3: Gemcitabine-induced recruitment of XPF to chromatin depends on APE. HCT116, APE-suppressed HCT116 (HCT116 shAPE), and XPF-deficient HCT116 (HCT116 g4-10) cells were treated with 1 µM of gemcitabine and chromatin fractions were isolated in the indicated time. The presence of XPF and APE was detected by western blots. The asterisk (*) shows a cross-reacted protein with the anti-XPF antibody. Histone H2AX was used as a loading control. Gemcitabine-induced recruitment of XPF to chromatin (lanes 1-3) was compromised in HCT116 shAPE cell line (lanes 4-5). The chromatin-bound APE is not changed by the gemcitabine treatment (lanes 4-6, Supplementary Figure 5). A signal of XPF (and APE) was normalized with a signal of H2AX in each chromatin fraction using ImageJ software. Then a ratio of chromatin-bound XPF with gemcitabine to XPF in control was determined and depicted as bar graphs. Three independent experiments were performed and averages of the ratio at indicated time points were plotted. The error bars show standard deviations. Only the difference in the chromatin-bound XPF between chromatin from control experiments and chromatin that was incubated one hour with 1 µM gemcitabine is statistically significant in HCT116 (p<0.05).