Topo II inhibitor ICRF-193 increases Topo IIα SUMOylation at CTD and up-regulates SUMOylation on mitotic centromeres and chromosome arms in XEEs. (A) Schematic representation for the primary structure of WT X. laevis Topo IIα and Topo IIα 3KR mutant. The three lysine residues indicated in the CTD were mutated to arginine, which inhibits SUMO2/3 conjugation of the CTD. (B) Endogenous Topo IIα in CSF XEEs was depleted using affinity-purified anti-Topo IIα antibody and replaced with recombinant full-length WT T7-tagged Topo IIα or Topo IIα 3KR (left). β-Tubulin, loading control for Topo IIα levels in CSF XEEs. The inhibitor-treated mitotic chromosomes were isolated from Topo IIα–replaced CSF XEEs and probed for Topo IIα SUMOylation using T7 antibody by Western blotting (right). Histone H4 is the loading control for the mitotic chromosomes. (C) DMSO- and ICRF-193–treated mitotic replicated chromosomes were isolated from XEEs as shown in Fig. 1 A with or without dnUbc9 (control). The mitotic chromosomes were subjected to immunofluorescence staining using the indicated antibodies, and DNA was stained with Hoechst 33342. Bars, 10 µm. (D) Quantification of mitotic chromosomes showing arm region SUMO2/3 signals. The mitotic chromosomes with arm region SUMO2/3 signal were counted for 30 chromosomes from three independent experiments (n = 3). Error bars, standard deviation. *, P value from Student’s t test. ***, Statistically significant difference, P ≤ 0.001.