Fig 1.

Expression and localization of meiotic cohesin components in ESCs. a Cohesin complexes in mitosis and meiosis. The cohesin complex is composed of long coiled-coil SMC subunits, referred to as SMC3 and SMC1α for mitotic cohesins, and SMC3 and SMC1β for meiotic cohesins [20, 21]. SMC complexes combined with the α-kleisin subunits (RAD21 for mitosis and REC8/RAD21L for meiosis) form a V-shaped structure [20, 21]. Cohesin subunits also include STAG1/2 for mitotic cohesin and STAG3 for meiotic cohesin, as well as PDS5A/B, Sororin, and WAPL, all of which are directly involved in the formation of cohesin complexes. b Expression analysis of cohesin components. Whole-cell lysate samples were extracted from asynchronous MEFs, ESCs (J1), and testis of C57BL/6J mice. α-tubulin was used as a loading control. The arrowhead indicates a non-specific band. c Comparison of expression level of SMC3, RAD21, SMC1α, STAG1, STAG2, REC8, RAD21L, STAG3, and SMC1β between ESCs and MEF, as assessed by (i) western blotting and (ii) qPCR analyses. d Immunoprecipitation (IP) analysis in MEFs and ESCs. Cohesin subunits were pulled down the using anti-cohesin (SMC3, RAD21, REC8, STAG1, STAG2, and STAG3) antibodies from whole-cell extracts in MEFs and ESCs. e,f Representative images of the interphase FISH analysis for the detection of sister chromatid separation in ESCs and MEFs. The signals of the locus-specific probe, bound to the arm regions (Chromosome 4: 116,094,264–116,123,690), are shown in red and DAPI-counterstained chromosomes are shown in blue. Scale bars = 2.5 μm. g,h Quantification of spots in each nucleus. The error bars represent the mean ± SD value from three biological replicates (n ≥ 50 for condition). i Physical interaction between REC8 and STAG3 in ESCs as demonstrated by IP analysis. j Quantification of REC8 intensity in the siCtrl, siSTAG3, and STAG3 expression vector (pSTAG3) (n = 130). Each biological replicate is color-coded (red, green, and blue) 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 three independent biological replicates. a.u., arbitrary unit. P-values (paired two-tailed t-test) were calculated using GraphPad Prism 5 software; **P < 0.01, ***P < 0.001. k Analysis of REC8 localization in the presence or absence of STAG3. α-tubulin and Lamin B were used as cytoplasmic and nuclear protein loading markers, respectively. pSTAG3, STAG3 expression vector; N, nucleus; C, cytoplasm. l Quantification of REC8 levels in cytoplasm and nucleus. The error bars are the mean ± SD from the biological replicates. m STAG3 binds directly to REC8 and maintains stabilization of REC8. The REC8–STAG3 complexes translocate into the nucleus by direct physical interaction