Figure S4.
Top2 inhibition does not accelerate midbody disassembly in normally segregating cells. (A–C) Localization and mean intensity of phosphorylated ATM-S1981 (pATM) at the midbody in normally segregating BE cells, in the absence (control) or presence of Top2α siRNA. Tubulin values indicate midbody thickness. Mean ± SD from n cells. Values in control were set to 1. (D–F) Time-lapse microscopy analysis of HeLa cells expressing tubulin:GFP. Cells were untreated (control) or treated with 10 μM ICRF-193 immediately before filming. Midbodies are shown by solid arrows. Time is from midbody formation to midbody disassembly (dotted arrows). (G and H) Western blot analysis of total Top2α, GFP, and tubulin in BE cells expressing siRNA-resistant (R) WT, Y805F, or AEEA GFP:Top2R, in the absence or presence of Top2α siRNA. (I) Mean intensity of TUNEL staining at the bridge DNA next to the midbody in cells expressing WT, Y805F, or AEEA GFP:Top2, in the presence of Top2α siRNA. Mean ± SD from n cells. Values in WT were set to 1. Numbers below/next to each bar indicate n. (J) Frequency of interphase cells exhibiting >10 γ-H2AX foci per nucleus after treatment with 10 μM MG132 and/or 10 μM etoposide for 4 h, in the absence or presence of ZNF451:GFP. Mean ± SD from three independent experiments (n > 300). ***, P < 0.001 (ANOVA and Student’s t test). (K and L) Localization of γ-H2AX or MDC1 in cytokinesis with chromatin bridges. (M) Western blot analysis of total Mre11, Rad17, and actin. (N and O) Localization of RFC3 or RFC5 in BE cells with chromatin bridges. Arrowheads indicate DNA knots. Midbodies (boxed areas) were reexported and are shown at higher magnification. Scale bars, 5 μm. Source data are available for this figure: SourceData FS4.

Top2 inhibition does not accelerate midbody disassembly in normally segregating cells. (A–C) Localization and mean intensity of phosphorylated ATM-S1981 (pATM) at the midbody in normally segregating BE cells, in the absence (control) or presence of Top2α siRNA. Tubulin values indicate midbody thickness. Mean ± SD from n cells. Values in control were set to 1. (D–F) Time-lapse microscopy analysis of HeLa cells expressing tubulin:GFP. Cells were untreated (control) or treated with 10 μM ICRF-193 immediately before filming. Midbodies are shown by solid arrows. Time is from midbody formation to midbody disassembly (dotted arrows). (G and H) Western blot analysis of total Top2α, GFP, and tubulin in BE cells expressing siRNA-resistant (R) WT, Y805F, or AEEA GFP:Top2R, in the absence or presence of Top2α siRNA. (I) Mean intensity of TUNEL staining at the bridge DNA next to the midbody in cells expressing WT, Y805F, or AEEA GFP:Top2, in the presence of Top2α siRNA. Mean ± SD from n cells. Values in WT were set to 1. Numbers below/next to each bar indicate n. (J) Frequency of interphase cells exhibiting >10 γ-H2AX foci per nucleus after treatment with 10 μM MG132 and/or 10 μM etoposide for 4 h, in the absence or presence of ZNF451:GFP. Mean ± SD from three independent experiments (n > 300). ***, P < 0.001 (ANOVA and Student’s t test). (K and L) Localization of γ-H2AX or MDC1 in cytokinesis with chromatin bridges. (M) Western blot analysis of total Mre11, Rad17, and actin. (N and O) Localization of RFC3 or RFC5 in BE cells with chromatin bridges. Arrowheads indicate DNA knots. Midbodies (boxed areas) were reexported and are shown at higher magnification. Scale bars, 5 μm. Source data are available for this figure: SourceData FS4.

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