Fig. 3

SMC4 expression changes in the cytosol and nucleus after SMC3 knockdown. (A) SMC3 protein expression in ES cells was suppressed by transfection with an siRNA pool against SMC3 (siSmc3). Cell transfected with 100 nM non-targeting RNA (siCtrl) were included. (B) The level of SMC3 relative to α-tubulin was normalize to the siRNA-transfected cell. (C) The cell-cycle profiles of ES cells treated with siSmc3 as characterized using FACS analysis. (D) Quantitative analysis of (C) in response to SMC3 knockdown. The percentages of siCtrl-transfected and SMC3-deficient cells in G1, S, and G2-phase were quantified using FlowJo software. Three independent experiments were performed. Error bars denote the mean ± SD (n = 3). (E) IP analysis for physical interaction between CTCF, cohesin, and condensin components. (F) Expression analysis of cohesin and condensin components in the presence or absence of SMC3. α-tubulin and Lamin B were used as cytoplasmic and nuclear protein loading markers, respectively. (G) Quantification of SMC3, SMC4, and RAD21 levels in cytoplasm and nucleus; Cy, cytosol; Nu, nucleus. The error bars are the mean ± SD from the biological replicates. (H) Analysis of SMC4 expression pattern during cell cycle progression. After a DTB, cells were released for cell cycle progression as shown in (Fig. 2B). α-tubulin and Lamin B were used as cytosol and nucleus markers, respectively. (I) Quantification of SMC4 expression levels of (H). The error bars denote the mean ± SD from three independent biological replicates.