Ti e super research live cell imaging system

Cells were fixed with 4% PFA for 5 min and permeabilized with 0.2% Triton X-100 for 2 min and then blocked in 1% BSA for 30 min at 37°C. Antibody dilutions were prepared in 1% BSA. Slides were counterstained with DAPI.
Three-dimensional datasets were acquired using a wide-field microscope (NIKON Ti-E super research Live Cell imaging system) with a NA 1.45 Plan Apochromat lens. The datasets were deconvolved with NIS Elements AR analysis software (NIKON). Three-dimensional datasets were converted to Maximum Projection in the NIS software, exported as TIFF files and imported into Adobe Photoshop for final presentation.
For the quantification of lamin A/C-S22ph, three-dimensional stack images were acquired with the same exposure. Using NIS-Element AR, an area corresponding to chromosomes or the nuclei was used to measure the mean intensity of lamina S22ph signals. For the total lamin A/C-S22ph, an area containing all lamin A/C S22ph signals was selected and used to measure mean intensity of total lamin A/C-S22ph. Areas of the same size within each image were used to identify and subtract the background from the measurements.
For the quantification of the nuclear circularity, the NIS Elements AR analysis software (NIKON) was used.
Violin plots were generated using the ggplot2 package in R.

Cells were fixed in 4% PFA and processed as previously described26 (link). Primary antibodies were used as in Supplementary Table 1. Fluorescence-labelled secondary antibodies were applied at 1:200 (Jackson ImmunoResearch). Three-dimensional data sets were acquired using a wide-field microscope (NIKON Ti-E super research Live Cell imaging system) with a numerical aperture (NA) 1.45 Plan Apochromat lens. The data sets were deconvolved with NIS Elements AR analysis software (NIKON). Three-dimensional data sets were converted to Maximum Projection in the NIS software, exported as TIFF files, and imported into Adobe Photoshop for final presentation.
Live cell imaging was performed with a Nikon Ti-E super research Live Cell imaging system microscope as previously described9 (link).
For quantification of the staining in RNAi background masks were created around the DAPI nuclei. Mean intensity of antibodies signals were extracted and background was subtracted. For quantification of enrichment in LacI/LacO systems, three circles were designed around the LacI spot, within the nucleus and outside the cell and signals intensities were extracted. The outside circle served as background and was subtracted from both the nuclear and the LacI spot, then the signal intensity from the LacI was normalized relative to the intensity of the nuclear signal.

Live cell imaging for Fig. 2E was performed with a DeltaVision microscope as previously described (Vagnarelli et al., 2011 (link)).
For Fig. 4G,H and Fig. S2, HeLa cells were seeded onto Labtech chamber slides with complete Leibovitz's L-15 medium, lacking phenol red (Gibco). Cells were imaged by differential interference contrast microscopy with a 20× objective (NA 0.45) and a wide-field microscope (NIKON Ti-E super research Live Cell imaging system) at 37°C. Images were captured every 30 min over a period of 24.5 h. Analyses of mitotic progression were conducted manually.