PR-619 induces TOP2 complexes in K562 cells with similar efficiency as etoposide, and complex formation is not dependent on ubiquitination. Cells were treated with PR-619, etoposide, or solvent control. Where indicated, cells were also treated with the E1 UEA inhibitor MNL7243 (10 μM) prior to adding etoposide or PR-619. Cells were embedded in agarose on microscope slides and processed by TARDIS analysis for TOP2A, TOP2B, and ubiquitin. Bar graphs represent the mean of the median values derived from replicates, which are also indicated individually as blue-lined circles. Error bars represent the S.D. MNL7243 pretreatment was for 2 hours. Statistical analysis was performed using t tests. For the last column in the left and right panel highlighted ($ for the PR-619 concentration), cells were treated with 80 μM PR-619 and processed with other samples, but with the omission of the primary antibody during immunofluorescence, to test for autofluorescence originating in the sample.