Live mitotic HeLa cell treated with epsin1 siRNA, DiOC6(3)to label mitotic membranes (green), and Hoechst 33258 to label chromosomes (blue). Confocal images were taken at 0.118 μm steps along the Z-axis. Epsin1 depleted cells show aberrant membrane morphologies compared with control cells (see grouped image).

Live mitotic HeLa cell treated with control siRNA, DiOC6(3)to label mitotic membranes (green), and Hoechst 33258 to label chromosomes (blue). Confocal images were taken at 0.118 μm steps along the Z-axis.

To visualize if HiLands-B LADs indeed move away from the NL upon lamin loss, we selected two regions consisting of mostly HiLands-B and exhibiting decreased emerin DamID values upon lamin loss for FISH analyses using Oligopaint. We used FISH to label the HiLands-B LADs while immunostained on Emerin to mark the nuclear periphery. Using Imaris, we quantified the distance between the FISH signal and the NL and found that the two HiLands-B regions in TKO mESCs were farther away from the NL than in the WT.

To visualize whether HiLands-P chromatin is decondensed, we used Oligopaint method -a modified fluorescence in situ hybridisation (FISH) technique specifically for imaging large Chromatic loci-to quantify four selected HiLands-P regions from chromosomes 1, 4, 13, and 14. After the hybridisation, we used Imaris software to quantify both the volumes and surface areas of these FISH signal in WT and TKO mESCs. The comparison demonstrated that both the volumes and surface areas of these regions increased significantly in TKO mESCs.