CDA prevents supernumerary UFB formation in BS cells and in cells expressing BLM.

<p>(A) Upper panel: Representative immunofluorescence deconvoluted z‐projection images of PICH-positive UFBs in BS-Ctrl_(BLM) and in BS-BLM anaphase cells. DNA was visualized by DAPI staining (blue). Centromeres were stained with CREST serum (in magenta) and UFBs were stained with PICH antibody (in green). In the enlarged image, CREST foci at the extremities of the UFB are indicated by yellow arrows. Scale bar: 5 μm. Lower panel: Fluorescence quantifications along UFBs using Image J. CREST-positive UFBs are characterized by CREST fluorescence signal at the extremities (left). CREST-negative UFBs present no CREST signal (right). (B) Mean number of UFBs (black bars) and chromatin bridges (gray bars) per anaphase cell in BS-Ctrl_(BLM), BS-BLM, BS-Ctrl_(CDA) and BS-CDA cell lines (upper panel); BLM and CDA levels, assessed by immunoblotting (lower panel). Errors bars represent means ± SD from five independent experiments (> 170 anaphase cells per condition (UFBs) or > 95 anaphase cells per condition (chromatin bridges)). (C) Mean number of UFBs (black bars) and of chromatin bridges (gray bars) per anaphase cell in BS-Ctrl_(CDA) cells and in BS-BLM-CDA cells transiently transfected with the indicated siRNAs (left panel); BLM and CDA levels, assessed by immunoblotting (right panel). Error bars represent means ± SD from three independent experiments (> 120 anaphase cells per condition (UFBs) or > 70 anaphase cells per condition (chromatin bridges)). (D-E) Relative numbers of UFBs (black bars) and of chromatin bridges (gray bars) per anaphase cell in BS-Ctrl_(BLM) and BS-BLM cell lines left untreated or treated with (D) 100 μM tetrahydrouridine (THU) or (E) 100 μM deoxyuridine (dU). Error bars represent means ± SD from: (D) five independent experiments (> 140 anaphase cells per condition (UFBs) or > 95 anaphase cells per condition (chromatin bridges)), (E) five independent experiments (> 120 anaphase cells per condition (UFBs) or > 95 anaphase cells per condition (chromatin bridges)). (F) Mean number of UFBs per cell (left panel), and percentage of chromatin bridge-positive anaphase cells (right panel) in HeLa-Ctrl_(CDA) and HeLa-shCDA cells. Error bars represent means ± SD from six independent experiments (> 155 anaphase cells per condition (UFBs) or > 100 anaphase cells per condition (chromatin bridges)). The significance of differences was assessed with Student’s <i>t</i>-test.