Extended Data Figure 8: Fluorescence correlation spectroscopy of endogenous Ki-67 tagged with EGFP.

a, Western blot performed on whole cell lysates of a HeLa cell line overexpressing GFP-tagged Ki-67 from a bacterial artificial chromosome (BAC), and of two different clones in which all endogenous Ki-67 alleles were N-terminally tagged with EGFP (C1, C2). The two lower bands labelled by anti-Ki-67 antibody in the BAC cell line (black arrows) correspond to the two wild-type Ki-67 isoforms. The EGFP–Ki-67 versions appear upshifted (green arrows) and the band of the small EGFP-tagged isoform overlaps with the band of the wild-type large isoform. b–h, FCS measurement from 3 independent experiments with 111 cells (C1) and 156 cells (C2). b, Imaging and analysis pipeline for FCS-calibrated imaging. Metaphase cells were identified based on Hoechst staining and imaged in 3D (only the central slice is shown). In the cytoplasm of the central slice an FCS measurement was performed. From the photon counts (right upper panel) the autocorrelation function (ACF) was computed (right lower panel). From the fit of the ACF to equation S1 in the Supplementary Information, the number of particles N in the focal volume was obtained. The concentration at the FCS point was computed by dividing N by the effective focal volume V_eff and the Avogadro’s constant N_A. For details see Supplementary Methods. c, A calibration curve was obtained by plotting the concentration computed from FCS against the fluorescence intensity in a 5 × 5 pixel region at the FCS measurement point (equation S2 in the Supplementary Information, dashed line). d, Fluorescence microscopy image of live HeLa cell with endogenous Ki-67, labelled by EFGP, scaled to absolute Ki-67 concentration as determined by FCS. e, Example segmentation of chromosomes and the chromosome surface used for quantification in f–h. Segmentation was performed in 3D, but for simplification only a single z-section is shown. A chromatin mask was obtained by segmentation of the Hoechst signal. To determine Ki-67 concentration on chromosomes, the mask was dilated to include Ki-67 signal at the outer chromosome surface (expanded chromatin mask). To calculate Ki-67 molecules on the outer chromosome surface, a rim around the chromatin mask was used (outer rim mask). See Supplementary Methods for details. f–h, Quantification of FCS measurements. Boxes indicate median, quartiles and 1.5 × interquartile range. f, Mean Ki-67 concentration in the cytoplasm and on 3D-segmented chromosomes using the expanded chromatin mask depicted in e. g, Total number of Ki-67 molecules within the whole DNA volume using the expanded chromatin mask depicted in e. h, Density of Ki-67 molecules on the chromosome surface using the outer rim mask depicted in e. Scale bars, 10 μm.