Figure S5.
Dicentric chromatin bridges exhibit impaired formation of double-strand DNA ends at the bridge DNA next to the midbody. (A) Localization of RFC4 in BE cells with chromatin bridges. (B) Mean intensity of Rad17 at the bridge DNA next to the midbody on chromatin bridges of various lengths. Values in <20 μM were set to 1. (C) BrdU-associated fluorescence in cytokinesis with chromatin bridges. Midbodies (boxed areas) were reexported and are shown at higher magnification. (D) Mean intensity of Mre11 at the bridge DNA next to the midbody in cells induced by HU, in the absence (control) or presence of Rad17 siRNA. (E) Mean intensity of Aurora B at the midbody ring. Mean ± SD from n cells. Values in control were set to 1. (F) Frequency of broken chromatin bridges in cytokinesis. Mean ± SD from three independent experiments (n > 120). (G and H) Western blot analysis of total Rad17, ATM, INCENP, Mre11, Aurora B, Top2α, GFP, and tubulin in cells transfected with Rad17 or Top2α siRNA, and/or expressing the siRNA-resistant GFP:Top2(Y805F)R:Rad17Δ. (I) Μean intensity of GFP at the bridge DNA next to the midbody in BE cells expressing siRNA-resistant (R) WT, Y805F, or GFP:Top2(Y805F)R:Rad1Δ in the presence of Top2α siRNA. Mean ± SD from n cells. Values in WT were set to 1. (J) Schematic of a potential mechanism to generate dicentric chromosome bridges by fusion of chromatids from different chromosomes in RPE-1 cells after induction with TC. Deprotected tel sequences are shown in green. (K) FISH analysis of HU-induced chromatin bridges in RPE-1 cells using a tel probe. The DNA bridge (boxed area) is shown at higher magnification. (L) Frequency of HU-induced chromatin bridges that are positive for cen or tel probes by FISH. Mean ± SD from three independent experiments (n > 60). (M) Mean intensity of TUNEL staining at the bridge DNA next to the midbody in HU- or TC-induced cells. Mean ± SD from n cells. Values in HU-induced were set to 1. ***, P < 0.001 (Student’s t test). Numbers below/next to each bar indicate n. (N) Experimental procedure to generate dicentric bridges with DNA knots in RPE-1 cells. (O) Schematic of a potential mechanism that generates dicentric bridges with DNA knots. (P) FISH analysis of TC+HU-induced chromatin bridges using a tel probe. A tel-positive signal on the DNA bridge is indicated by an arrow and shown at higher magnification. Arrowheads indicate DNA knots. (Q) Frequency of TC+HU-induced chromatin bridges that are positive for tel probe by FISH. Mean ± SD from three independent experiments (n > 60). Scale bars, 5 μm. Source data are available for this figure: SourceData FS5.

Dicentric chromatin bridges exhibit impaired formation of double-strand DNA ends at the bridge DNA next to the midbody. (A) Localization of RFC4 in BE cells with chromatin bridges. (B) Mean intensity of Rad17 at the bridge DNA next to the midbody on chromatin bridges of various lengths. Values in <20 μM were set to 1. (C) BrdU-associated fluorescence in cytokinesis with chromatin bridges. Midbodies (boxed areas) were reexported and are shown at higher magnification. (D) Mean intensity of Mre11 at the bridge DNA next to the midbody in cells induced by HU, in the absence (control) or presence of Rad17 siRNA. (E) Mean intensity of Aurora B at the midbody ring. Mean ± SD from n cells. Values in control were set to 1. (F) Frequency of broken chromatin bridges in cytokinesis. Mean ± SD from three independent experiments (n > 120). (G and H) Western blot analysis of total Rad17, ATM, INCENP, Mre11, Aurora B, Top2α, GFP, and tubulin in cells transfected with Rad17 or Top2α siRNA, and/or expressing the siRNA-resistant GFP:Top2(Y805F)R:Rad17Δ. (I) Μean intensity of GFP at the bridge DNA next to the midbody in BE cells expressing siRNA-resistant (R) WT, Y805F, or GFP:Top2(Y805F)R:Rad1Δ in the presence of Top2α siRNA. Mean ± SD from n cells. Values in WT were set to 1. (J) Schematic of a potential mechanism to generate dicentric chromosome bridges by fusion of chromatids from different chromosomes in RPE-1 cells after induction with TC. Deprotected tel sequences are shown in green. (K) FISH analysis of HU-induced chromatin bridges in RPE-1 cells using a tel probe. The DNA bridge (boxed area) is shown at higher magnification. (L) Frequency of HU-induced chromatin bridges that are positive for cen or tel probes by FISH. Mean ± SD from three independent experiments (n > 60). (M) Mean intensity of TUNEL staining at the bridge DNA next to the midbody in HU- or TC-induced cells. Mean ± SD from n cells. Values in HU-induced were set to 1. ***, P < 0.001 (Student’s t test). Numbers below/next to each bar indicate n. (N) Experimental procedure to generate dicentric bridges with DNA knots in RPE-1 cells. (O) Schematic of a potential mechanism that generates dicentric bridges with DNA knots. (P) FISH analysis of TC+HU-induced chromatin bridges using a tel probe. A tel-positive signal on the DNA bridge is indicated by an arrow and shown at higher magnification. Arrowheads indicate DNA knots. (Q) Frequency of TC+HU-induced chromatin bridges that are positive for tel probe by FISH. Mean ± SD from three independent experiments (n > 60). Scale bars, 5 μm. Source data are available for this figure: SourceData FS5.

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