Journal: bioRxiv

Article Title: MYC modulates TOP2A diffusion to promote substrate detection and activity

doi: 10.1101/2025.07.15.665031

Figure Lengend Snippet: a , STED images of HCT116 cells immunostained for TOP2A, RNAPI subunit RPA194, nucleolar proteins Fibrillarin (FIB) and Nucleophosmin (NPM). Two independent experiments were performed. Scale bar = 5 μm. b , Confocal images of HCT116 cells treated with CSK buffer to remove soluble proteins +/− RNAse immunostained for TOP2A and FIB, and DNA stained with DAPI. Four independent experiments were performed. Scale bar = 5 μm. c , Representative STED image of HCT116 cell (left) immunostained for TOP2A and Mediator component MED4. Average MED4 (middle) and TOP2A (right) signal at 8,240 MED4 puncta from 30 cells from three independent experiments. Scale bar as indicated. d , STED images of HCT116 cells treated with TOP2 inhibitor 5 μM ICRF-193 for 30 minutes or 5 nM Actinomycin D (ActD) for 1 hour to specifically inhibit RNAPI, immunostained for TOP2A, and FIB, and DNA stained with DAPI. Two independent experiments were performed. Scale bar = 5 μm. e, Quantitation of nucleolar:nucleoplasmic ratio of TOP2A signal normalized to untreated control (CTRL) after treating HCT116 cells (left) with 5 μM ICRF-193 for 15 minutes or HCT116 TOP1-AID cells (right) with 500 μΜ auxin for 1 hour, to degrade TOP1. Six independent experiments were performed. 550 CTRL cells or 604 ICRF-treated cells (left) and 732 CTRL cells or 889 auxin-treated cells (right) were measured. ****p < 0.0001 (unpaired t test). f , Nuclear γH2A.X intensity of HCT116 TOP1-AID cells +/− 500 μM auxin for 1 hour +/– 1 μM etoposide (Eto) treatment for 1 hour, normalized to respective “– Eto” conditions. Eight independent experiments were performed. 1020 CTRL cells, 1085 Eto-treated cells, 1188 auxin-treated cells, or 1378 auxin and Eto-treated cells, were measured. ****p < 0.0001 (Ordinary one-way ANOVA, Šidák correction).

Article Snippet: Finally, coverslips were washed three times in PBS-T and mounted using ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher P36966) and sealed with nail polish or mounted using ProLong Diamond Antifade Mountant (Thermo Fisher P36970) and left to cure overnight.

Techniques: Staining, Quantitation Assay, Control

Journal: bioRxiv

Article Title: MYC modulates TOP2A diffusion to promote substrate detection and activity

doi: 10.1101/2025.07.15.665031

Figure Lengend Snippet: a , Representative confocal images of HCT116 TOP1-AID cells +/− auxin treatment immunostained for TOP2A and FIB, and DNA labelled with DAPI. Scale bar = 5 μm. Quantitated in . b , Representative confocal images of HCT116 cells +/− ICRF-193 treatment immunostained for TOP2A and FIB, and DNA labelled with DAPI. Scale bar = 5 μm. Quantitated in . c , d , Representative images of HCT116 TOP1-AID cells +/− auxin and +/− Eto treatment immunostained for γH2AX, TOP1 (A) or FIB (B) and DNA labelled with DAPI. Quantitated in . e , Quantitation for experiment in d , where the γH2AX signal is separated into nucleolar and nucleoplasmic based on overlap with nucleolar marker FIB. 598 CTRL untreated cells, 576 CTRL Eto-treated cells, 566 auxin untreated cells, and 632 auxin Eto-treated cells were measured. ****p < 0.0001 (ordinary one-way ANOVA with Šidák correction). f , Representative images of HCT116 cells +/− ICRF-193 treatment immunostained for TOP2B and FIB, and DNA labelled with DAPI. g , Quantitation of nucleolar:nucleoplasmic ratio of TOP2B signal in HCT116 cells treated +/− ICRF. Four independent experiments were performed, normalized to respective CTRL conditions. 327 CTRL cells and 425 ICRF-treated cells were measured. **p < 0.01 (unpaired t-test).

Article Snippet: Finally, coverslips were washed three times in PBS-T and mounted using ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher P36966) and sealed with nail polish or mounted using ProLong Diamond Antifade Mountant (Thermo Fisher P36970) and left to cure overnight.

Techniques: Quantitation Assay, Marker

Journal: bioRxiv

Article Title: MYC modulates TOP2A diffusion to promote substrate detection and activity

doi: 10.1101/2025.07.15.665031

Figure Lengend Snippet: a , Representative images of HCT116 cells immunostained for MYC and FIB, and DNA labelled with DAPI. b - d , Diffusion coefficients of Fast ( b ), Slow ( c ), and Bound ( d ) nuclear fractions from individual Halo-TOP2A HCT116 MYC-AID cells treated with control, auxin or triptolide (5 μM for 60 minutes). 110-129 cells for each condition from three independent experiments. ****p < 0.0001; **p < 0.01; *p < 0.05 (ANOVA, Dunnett’s multiple comparison test).

Article Snippet: Finally, coverslips were washed three times in PBS-T and mounted using ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher P36966) and sealed with nail polish or mounted using ProLong Diamond Antifade Mountant (Thermo Fisher P36970) and left to cure overnight.

Techniques: Diffusion-based Assay, Control, Comparison