Fig. 3

Excessively loaded cohesin are anchored by CTCF and bow-tie topologically associating domains. A Super-resolution images of H2B, CTCF, NIPBL, MAU2 in the presence of RAD21 over-expression labelled with antibodies. Scale bar, 5 μm. 3 independent experiments. Deconvolution was applied to improve the imaging resolution and signal to noise ratio. B Co-localization analysis of vermicelli and protein in (A) (n ≈ 40 per condition). The lower quartile, median and upper quartile values were labelled in the box. (***P < 0.001, two sample T test). C Co-localization ratio of CTCF and RAD21 in the presence of RAD21 over-expression (n = 54 per condition). (***P < 0.001, two sample T test). D Super-resolution images of EMC7 TAD in the absence or presence of RAD21 over-expression labelled by in situ hybridization with DNA probes, RAD21 was labelled with antibodies. Scale bar, 5 μm. Boxed regions are shown as magnified inserts. Scale bar, 1 µm. 2 independent experiments. Deconvolution was applied to improve the imaging resolution and signal to noise ratio. E The normalized intensity profile of boxed region in G measured across the center of the EMC7 TAD and RAD21 (n = 20 per condition). F Quantification of volume change of EMC7 TADs after RAD21 over-expression. (another assay without RAD21 labelled, n > 380 per condition). The lower quartile, median and upper quartile values were labelled in the box. (***P < 0.001, two sample T test). G Scheme of proposed cohesin-mediated loop model in the absence and presence of RAD up-regulation. H Example images of chromosome territory (CT) 2 in RAD21 over-expressed HeLa cell obtained by CT2 probes, JF549 and DAPI. Scale bar, 5 μm. 2 independent experiments. Deconvolution was applied to improve the imaging resolution and signal to noise ratio. I Quantification of morphology change of CT2 by calculating the Radius of gyration (n = 77 per condition). (***P < 0.001, two sample T test)