Fig 2: Overexpression of ORC2-SUMO2 fusion protein partially reversed phenotype of cells stably expressing PIAS4 shRNA or SENP2(A) Various U2OS cells lines as indicated were subjected to western blot analysis with indicated antibodies. (B) 2X105 U2OS cells stably expressing control shRNA, control shRNA/ORC2-SUMO2, PIAS4 shRNA or PIAS4 shRNA/ORC2-SUMO2 as indicated (upper panel) or EV, EV/ORC2-SUMO2, SENP2 or SENP2/ORC2-SUMO2 as indicated (lower panel) were plated and cell number was counted every day for five days. The graph shows the average of three independent experiments; mean ± SD. *, p < 0.05; **, p < 0.01. (C and D) 1000 U2OS cells stably expressing control shRNA, PIAS4 shRNA or PIAS4 shRNA/ORC2-SUMO2 (C) or EV, SENP2 or SENP2/ORC2-SUMO2 (D) were seeded per well of a six-well plate. After incubation for 10–14 days, cells were stained with crystal violet (upper panel). Analysis of colony formation rates of cells (lower panel). Data are means ± SD of three independent experiments. *, p < 0.05; **, p < 0.01. (E) U2OS cells were transient transfected with Flag tagged ORC2-SUMO2. Western blot and FACS were performed to detect expression of Flag-ORC2-SUMO2 protein and cell cycle profile, respectively. (F and G) Cell cycle profiles of cells stably expressing control shRNA, PIAS4 shRNA or PIAS4 shRNA/ORC2-SUMO2 (F) or EV, SENP2 or SENP2/ORC2-SUMO2 (G) were analyzed by FACS.

Fig 3: Dynamic interaction between ORC2 and PIAS4 or SENP2(A) U2OS cells were treated with nocodazole for 24 hours and then released into fresh medium for indicated times. Immunoprecipitation was performed with ORC2 antibody and immunoprecipitates were blotted with ORC2 or PIAS4 antibody. Asterisks are indicated as non-specific bands and arrows are indicated as SUMOylated ORC2, non-SUMOylated ORC2, or IgG. (B) U2OS cells were treated with RO3306 and immunostaining was performed with PIAS4 antibody (green) and CENP-A antibody (red). Nuclei were stained with DAPI (blue). Scale bar: 10uM. (C) U2OS cells were treated with nocodazole for 24 hours and then released into fresh medium for indicated times as in (A). Immunoprecipitation was performed with ORC2 antibody and immunoprecipitates were blotted with ORC2 or SENP2 antibody. Asterisks are indicated as non-specific bands and arrows are indicated as SUMOylated ORC2, non-SUMOylated ORC2, or IgG. (D) U2OS cells were immunostained with SENP2 antibody. SENP2 staining (green) at different stages of mitosis was observed by microscope. Nuclei were stained with DAPI (blue). Scale bar: 10uM. (E) U2OS cells were transiently transfected with indicated plasmids. Cells were immunostained with HA antibody (green) and ORC2 antibody (red). Nuclei were stained with DAPI (blue). Scale bar: 10uM.

Fig 4: GSK3s promote the chromatin association of ESCO2 and SMC3 acetylation in S phase. (A) The outline of time-course experiments. The cells were synchronized using double-thymidine blocks. DMSO/GSK3 inhibitor was added half an hour before release. 5 h after release from the second thymidine block, nocodazole (Noc) was applied. Cell samples were harvested at different time points after DT Rel. (B) Chromatin fractions were prepared from 293T cells harvested as described above and analyzed by western blotting with the indicated antibodies. ORC2 served as a loading control of the chromatin-associated protein. (C) Quantification of SMC3 acetylation level in S phase (0–7 h after DT Rel). Band intensities in (B) were quantified by Quantity One software. The relative SMC3ac/SMC3 ratio was calculated. The statistical significance was analyzed via two-way ANOVA. The data represent mean ± SEM, **P > 0.01, ***P > 0.001. (D) Artificial dimerization of Eco1 rescues the cohesion defect caused by MCK1 deletion. Diploid yeast strains with the indicated genotypes (SBY214 background) were grown to log phase and sequentially synchronized with nocodazole in metaphase. Dimerization of Eco1 carrying FRB and FKBP tags was induced by adding rapamycin. At least 200 metaphase-arrested cells were counted for each experiment. Means ± SEM are shown. The statistical significance was calculated via one-way ANOVA, ****P< 0.0001.