Fig. 3
Chromosome compaction and precocious separation after the knockdown of cohesin components. a Metaphase chromosome images of telomere FISH from ESCs and MEFs expressing a non-targeting siRNA or different siRNAs against SMC3, RAD21, REC8, STAG3, and RAD21-REC8 (siSMC3, siRAD21, siREC8, siSTAG3, siRAD21-siREC8). Telomeric probe signals are shown in red and DAPI-counterstained chromosomes are shown in blue. Scale bars = 10 μm. b Metaphase chromosome images of diverse length. (i) Metaphase chromosomes stained with DAPI and telomere FISH. (ii) Measurement of chromosome length by telomere FISH. Scale bar = 2.5 μm. c Images of a metaphase spread from normal (left) and precocious separation (right) showing proper and defective chromosome separation, respectively. d Quantification of normal chromosomes and precocious separated chromosomes. The bar graph indicates the percentages of cells showing a ratio of normal chromosomes and precocious separated chromosomes among metaphase chromosomes (n = 200 per condition) from three biological replicates. The error bars represent the mean ± SD (n = 3). siR21, siRAD21; siR8, siREC8. e Quantification of chromosome length and percentage of average chromosome length per metaphase cells. Length of chromosomes was measured by calculating the distance between both sides of the telomere probes. Data are shown as mean ± SD value from at least 200 chromosomes per independent experiment (n = 3). f Compaction analysis of chromosomes from the prophase to the metaphase. ESCs expressing histone H2B-GPF were analyzed by fluorescence microscopy. Scale bars = 2.5 μm. g Representative images of ESCs marked with H2B-GFP in metaphase. h Quantification of chromosome compaction. Cell volume and intensity were analyzed using the Nikon NIS software. The X- and Y-axes of the graphs indicate intensity and volume (μm2), respectively. Each biological replicate is color-coded (red and green) and the average of each replicated data is indicated with a larger dot, and black bars indicate the averages of three means (n = 50 for condition). The error bars are the mean ± SD from two biological replicates. **P < 0.01, ***P < 0.001. i Metaphase spreads hybridized with the telomeric probe. The locus-specific probe (“Probe A”) binds to the region of Ch 4 (Chromosome 4: 116,094,264–116,123,690). Telomeric probe and locus-specific probe signals are shown in green and red, respectively. The lengths of chromosomes were measured by calculating the distance between the telomere and locus-specific probes. The chromosome lengths were analyzed using Nikon NIS software. Scale bars are 2.5 μm. j,k Quantification of chromosome lengths in metaphase chromosomes from cells treated or untreated with siRNA specific to the cohesin components for 24 h (n = 30 for condition). The error bars represent the mean ± SD from three independent experiments