(A) DAPI staining in control and stable Ki-67-knockdown U2OS cells. (B) HeLa cells stably expressing GFP-H2B and mCherry-H2B, depleted using Ki-67 or non-targeting (CTRL) siRNA. Left, FRET efficiency (cross shows mean value) ** Different, p<0.01. FRET efficiency and spatial distribution shown by a pseudocolour scale of FRET (%) values from 0 to 25%. Bars, 10 μm. (C) Left, representative HeLaH2B-2FP nuclei showing spatial distribution of FRET efficiency. Arrowheads show different chromatin compaction states (high FRET, red; intermediate, green; low, blue), Bars, 2 μm. Right, mean FRET distribution curves from siRNA control (blue curve, n=8) and siRNA Ki-67 (red curve, n=11) nuclei. (D) Relative fraction of the three FRET efficiency populations (blue (low), FRET efficiency ≤ 8%; green (medium), 8–15%; and red (high), 15–25%) in siRNA control and siRNA Ki-67 nuclei. Error bars indicate SD. (E) Immunofluorescence of CENP-A localisation in control and stable Ki-67 knockdown HeLa (left) and U2OS (right) cells. Nucleolar localisation (white arrows). Nucleolin was used as nucleolar marker. Bar, 10 µm. Below: quantification in different cells of numbers of CENP-A spots not associated with the nucleolus.