Fig 2: 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 SMC1a for mitotic cohesins, and SMC3 and SMC1ß for meiotic cohesins [20, 21]. SMC complexes combined with the a-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. a-tubulin was used as a loading control. The arrowhead indicates a non-specific band. c Comparison of expression level of SMC3, RAD21, SMC1a, STAG1, STAG2, REC8, RAD21L, STAG3, and SMC1ß 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. a-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

Fig 3: Knockdown of STAG2 or STAG3 impairs the effects of vemurafenib on inhibiting melanoma xenograft tumor growth in vivo(a) Nude mice bearing xenograft tumors of A375 cells stably expressing pTRIPZ-shSTAG2#60 were treated with vehicle, doxycyline (Dox), vemurafenib (Vem), or both doxycyline and vemurafenib. Unpaired two-tailed Student's t-test was performed to compare between two groups of mice that were treated with vemurafenib (mean ± s.e.m. * P < 0.05). The data variance is similar between groups. n = 5–7. (b) Waterfall plots showing the percent change in tumor volume at day 7 for the individual tumors in each treatment group of the STAG2 knockdown experiment. (c) Representative images of mouse tumor samples from the STAG2 knockdown experiment subjected for various immunohistochemical analyses as indicated. Scale bar: 50 µm. (d) Nude mice bearing xenograft tumors of A375 cells stably expressing pLKO-shSTAG3#69 were treated with control or vemurafenib (Vem) diet. Unpaired two-tailed Student's t-test was performed to compare between two groups of mice that were treated with vemurafenib (mean ± s.e.m. * P < 0.05). The data variance is similar between groups. n = 5–7. (e) Waterfall plots showing the percent change in tumor volume at day 8 for the individual tumors in each treatment group of the STAG3 knockdown experiment. (f) Representative images of mouse tumor samples from the STAG3 knockdown experiment subjected for various immunohistochemical analyses as indicated. Scale bar: 50 µm.

Fig 4: Immunoblot showing STAG2 expression in bladder tumour-derived cell lines. Proteins (30 µg) from 47 cell lines were analysed by western blot analysis using antibodies specific to STAG2. Asterisks denote cell lines carrying STAG2 mutations. Alpha-tubulin expression was used to assess protein loading.

Fig 5: USP13 interacts with soluble cohesin complexes preferentially during DNA replication.A, whole cell lysates (W), chromatin lysates (C), and soluble lysates (S) were prepared from 293T cells and Western blot was performed with antibodies to USP13, STAG2, tubulin (soluble protein), and histone H3 (chromatin protein). B, cohesin complexes were immunoprecipitated with SMC3 antibodies from the protein lysates described in A, and Western blot was performed with the antibodies indicated. C–D, 293T cells were treated for 20 h with dimethyl sulfoxide (DMSO) vehicle alone (DM); hydroxyurea (HU) to arrest cells in S phase; RO-3306 (RO) to arrest cells in G2; and nocodazole (NOC) to arrest cells in mitosis. C, flow cytometry was performed to document the cell cycle arrests. D, SMC3 immunoprecipitations were then performed on whole cell lysates, followed by Western blot with USP13 antibodies. Quantification of USP13 bands in SMC3 immunoprecipitates using ImageJ is shown.