Fig. 1: Restricted activation of UFB-binding complexes before anaphase onset.
From: Centromere protection requires strict mitotic inactivation of the Bloom syndrome helicase complex
a Co-immunoprecipitation in nocodazole/MG132-arrested RPE1 cells containing endogenous PICH-mAID-mClover3 ± PLK1i (100 nM). PLK1i was added at the last 1 hr. Relative IP ratios are shown. Repeated two and three experiments are shown in Supplementary Fig. 1a. b BLM, TOP3A and RIF1 at K-chromatin in MG132-arrested metaphase cells ± PLK1i (30 min). RPE1 endoPICH-mAID-mClover3 or HCT116 EGFP-TOP3A cells were co-stained for BLM; RPE1 EGFP-RIF1 cells were co-stained for PICH. Centromeres and K-chromatin are labelled by CREST and PICH, respectively. c BLM UFBs localisation in pre-anaphase and anaphase cells. Experimental outline (top). Representative images of pre-anaphase UFBs in SGO1-depleted cells and UFBs in control cells. Relative BLM intensities are shown. Data is normalised to average intensities of mid/late anaphase-UFBs (total number of UFBs from three independent experiments: siSGO1 pre-UFBs n = 586, 78 cells; early anaphase UFBs n = 555, 43 cells; mid/late anaphase UFBs n = 343, 62 cells; mean ± S.D. is shown). d similar in c but DMSO or PLK1i was added at the last 30 min. Representative images of BLM, PICH, and RPA on pre-anaphase UFBs. Maximum chromatid separation distances indicated by the yellow lines are shown. Arrowhead, non-disjoined chromatid arms. Numbers of mitotic cells analysed from three independent experiments: DMSO n = 56, 61, 64; PLK1i: n = 63, 66, 66; mean ± S.D. is shown. e Mitotic populations showing different sister-chromatid separation patterns in ICRF193-treated SGO1-depleted RPE1 cells ± PLK1i (30 min). Representative images are shown below the graph. Numbers of cells analysed from three independent experiments: DMSO n = 56, 61, 64; PLK1i: n = 63, 66, 66; mean ± S.D. is shown. f The PLK1i-induced anaphase-like population maintained high Cyclin B1 (numbers of cells analysed: pre-anaphase n = 78, anaphase n = 42 and anaphase-like n = 52; mean ± S.E.M. is shown). g Absence of midzone in the anaphase-like cells. INCENP was used as a midzone marker. Numbers of cells examined shown below the images. h PLK1 inactivation promotes anaphase-like separation of catenated chromosomes after sister-chromatid cohesion inactivation. Scale bars, 5 μm. All p values are calculated by unpaired two-tailed t-test. Source data are provided as a Source data file.
