Fig 2: MG132 inhibits the reversal of etoposide-induced TOP2A- and TOP2B-DNA complexes. (A) K562 cells were incubated with solvent, etoposide (100 µM), MG132 (50 µM) or were co-incubated with 50 µM MG132 and 100 µM etoposide for 2 h. After 2 h etoposide was removed, but MG132 was maintained in cell incubations that initially contained it. Levels of TOP2A and TOP2B DNA complexes at 0, 15, 30, 60 and 120 min after etoposide removal (wash-out) were determined using the TARDIS assay. Statistical comparisons were made between the levels of TOP2-DNA complexes in the presence or absence of MG132 by unpaired t-test. Figures are given below for cell viability (trypan blue exclusion) under the cell treatment conditions used: “-120 min” refers to the time at which drugs were first added to cells, “0” refers to the time at which etoposide wash-out was performed and “120 min” refers to 2 h post drug wash-out. (B) The treatments employed in (A) do not significantly affect cellular TOP2A or TOP2B levels. Cells were treated as indicated and fixed with paraformaldehyde on poly lysine-coated slides. Total TOP2A and TOP2B was quantified by immunofluorescence. 100 µM etoposide incubation did result in a small decrease in total cellular TOP2A. (C) Cell cycle distribution at time of etoposide wash-out (“0”) and 2 h after etoposide washout (“120”).

Fig 3: Etoposide inhibits the EMT/ß-catenin/STT3/PD-L1 axis through TOP2B degradation-dependent nuclear ß-catenin downregulation. a Effect of TOP2 isoforms knockdown on the expression of EMT markers, STT3 and PD-L1 in 4T1 cell. b Representative phase-contrast microscopy images of 4T1 cell treated with indicated sg-RNAs. Scale bar, 50 µm. c Influence of TOP2 isoforms knockdown on etoposide-induced MET and downregulation of STT3 and PD-L1. d Western blot analysis (left), PD-1 binding assay (upper right), and in vitro PBMC-mediated cancer cell killing assay (bottom right) of tumorspheres cultured from various sgRNA-treated 4T1 cells. e Effect of proteasome inhibitor (MG132) and caspase inhibitor (Z-VAD-FMK) on etoposide-induced TOP2B degradation, MET, and downregulation of STT3 and PD-L1. f Western blotting of whole-cell extracts analyzing the effect of etoposide and TOP2B knockdown on total and active (non-phospho) ß-catenin (ß-Cat). g Western blotting of whole-cell extracts (WCE), cytosolic (Cyto), membrane (Mem), or nuclear (Nuc) fractions from sgRNA-treated 4T1 cells analyzing the effect of TOP2B knockdown on ß-catenin subcellular localizations. Histone H3, E-cadherin (E-Cad), and tubulin were used as makers of nuclear, membrane, and cytosolic fractions, respectively. h Efficacy of ß-catenin knockdown (si-ß-Cat) in desensitizing cells to etoposide- and sg-TOP2B- induced MET and downregulation of STT3 and PD-L1. Nuc nuclear fraction, WCE whole-cell extracts. i Co-immunoprecipitation assay showing interaction between TOP2B and ß-catenin. j Duolink assay analyzing the interaction of TOP2B and active (non-phospho) ß-catenin in the nucleus. Red dots (indicated by arrows) represented TOP2B-ß-catenin interaction signals and nuclei were counterstained with DAPI (blue). The right column shows higher-magnification image of the area outlined in the left column. Scale bar, 5 µm. Error bars represent s.d. (n = 3). *P < 0.05, Student’s t-test. See also Supplementary Fig. 8