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Fig. 4 | Genome Biology

Fig. 4

From: Meiosis-specific cohesin complexes display essential and distinct roles in mitotic embryonic stem cell chromosomes

Fig. 4

Chromosome shortening during the metaphase upon the knockdown of cohesin components. a Venn diagram for identification of up- or downregulated genes from siRNA-mediated knockdown of SMC3, RAD21, and REC8 in ESCs. Data were adjusted with P < 0.1 and fold-change > 1.5. (Additional file 2: Table S1). b Comparison of gene expression of RB. RB transcript levels from qPCR and RNA sequencing experiments were analyzed to evaluate RB gene expression. The error bars are the mean ± SD (n = 3 for qPCR). The RB expression levels measured by RNA-Seq experiments are represented as average values of two biological replicates. c Analysis of RB protein levels following transfection with a siRNA pool against SMC3, REC8, RAD21, and STAG3 in ESCs. α-tubulin was used as a loading control. d Quantification of RB expression shown in c using Image Lab 6.0 software (Bio-Rad). Results are illustrated as mean ± SD value (n = 3). e Immunoprecipitation (IP) analysis for physical interaction between RB and CAP-D3 in ESCs. (i–iii) Western blotting; (iv) quantification of RB and CAP-D3 levels from IP results. The error bars are the mean ± SD from three independent biological replicates. f Immunofluorescence analysis of chromosome compaction in ESCs during the metaphase stage. Chromosomes were stained with anti-RB antibody, anti-CAP-D3, and DAPI. Ctrl, siControl; pRB, pEF1α-RB expression vector; siSMC3, siRAD21, siREC8, STAG3, and siCohesin/siRB1 indicate siRNA treatment against SMC3, RAD21, REC8, STAG3, and cohesin/RB1 respectively. g,h Intensity of RB and CAP-D3 protein signals per a chromosome in ESCs. a.u., arbitrary unit. The data are reported as mean ± SD values from three biological replicates (n > 100 for condition). Statistics: Paired two-tailed t-test; ns, not significant; ***P < 0.001

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