Fig. 6: RIF1 and PP1s facilitate the activation of UFB-binding complex.
From: Centromere protection requires strict mitotic inactivation of the Bloom syndrome helicase complex
a Experimental setup (top). Representative images and percentages of mitotic cells positive for BLM at K-chromatin and centromere unwinding are shown. Numbers of cells analysed from four independent experiments: RPE1 n = 73, 72, 68, 69; ΔRIF1 n = 70, 74, 74, 77; mean ± S.D. is shown. Western blotting shows RIF1 levels. b K-chromatin localisation of BLM in wildtype and ΔRIF1 RPE1 cells. Experimental setup (top). Representative images and percentages of metaphase-arrested cells positive for BLM at K-chromatin are shown. Numbers of cells analysed from three independent experiments: RPE1 DMSO n = 62, 58, 64; PLK1i n = 60, 60, 85; CDK1i n = 50, 50, 98; ΔRIF1: DMSO n = 54, 58, 63; PLK1i n = 50,77, 75; CDK1i n = 50, 60, 100; mean ± S.D. is shown. c Centromere unwinding in ΔRIF1 RPE1 cells with wildtype (WT) or PP1s-binding mutant (PP1bs) of EGFP-RIF1. Representative images and percentages of mitotic cells positive for centromere unwinding are shown. Numbers of cells analysed from three independent experiments: WT n = 70, 59, 88 and in PP1bs n = 70, 80, 84; mean ± S.D. is shown. d Centromere disintegration analysis. Representative images of metaphase chromosomes after PLK1 inhibition. Centromeres and telomeres are labelled by FISH PNA probes. Asterisks indicate chromatin with centromere breakage. Quantification of broken whole chromosome arms per metaphase spread. Numbers of metaphase spreads analysed from three independent experiments: RPE1 n = 31, 39, 73; ΔRIF1, n = 39, 33, 35; ΔRIF1 + EGFP-RIF1(WT) n = 37, 33, 41; ΔRIF1 EGFP-RIF1(PP1bs) n = 42, 39, 34. mean ± S.D. is shown. e Experimental outline of co-treatments of PP1s and PLK1 inhibitors. Representative images of BLM K-chromatin localisation in RPE1 metaphase cells. Graphs show quantification of average centromeric intensity of BLM relative to PICH per cell in RPE1 (left) and RPE1 EGFP-BLM (right) cells. Data is normalised to the average intensity in PLK1i condition. Numbers of cells analysed in each condition, RPE1: PLK1i n = 43; PLK1i + 1 μM PP1i n = 41; PLK1i + 5μM PP1i n = 40; RPE1 EGFP-BLM: PLK1i n = 29; PLK1i + 1μM PP1i n = 24; PLK1i + 5μM PP1i n = 17; mean ± S.E.M is shown. Scale bars, 5 µm. All p values are calculated by unpaired two-tailed t-test. Source data are provided as a Source data file.
