Fig. 5

Chromosome association of mitotic and meiotic cohesin components. a Representative images of prometaphase-like chromosomes in cohesin knockdown. ESC chromosomes synchronized with thymidine were stained with REC8 or RAD21 antibodies and DAPI. Scale bars = 2.5 μm. b Quantification of the intensity level of RAD21 and REC8 in ESC chromosomes. 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. ns: not significant, ***P < 0.001. c 3D structured illumination microscopy (SIM) images of RAD21 and REC8 in ESC chromosomes. d Representative images of prometaphase-like chromosomes in WAPL-knockdown ESCs. The synchronized cells were treated with siCtrl (Control, metaphase chromosome; first row) or siRNA specific to WAPL (second row), WAPL/RAD21 (third row), and WAPL/REC8 (fourth row). Chromosome spreads were stained them with REC8 or RAD21 antibodies and DAPI. e Quantification of cohesin intensity shown in d. Results are illustrated as mean ± SD value (n = 50 per condition). P-values (paired two-tailed t-test) were calculated using the GraphPad Prism 5 software: **P < 0.01, ***P < 0.001. f 3D SIM images of WAPL-knockdown cells. The fixed chromosomes were immunostained with REC8 and RAD21 antibodies. Scale bar = 2.5 μm. 3D SIM images were analyzed using NIS software from Nikon. Scale bar = 10 μm (Side view). g Fluorescence microscopy images of prometaphase-like chromosomes in WAPL-knockdown cells. Fixed chromosomes were immunostained with antibodies against RAD21, REC8, and SMC4. Scale bars = 2.5 μm. h Quantification of cohesin and condensin intensity. The error bars are the mean ± SD from three biological replicates (n ≥ 30 for condition). ***P < 0.001