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. 2022 Jan 26;50(17):9601–9620. doi: 10.1093/nar/gkac003

Figure 2.

Figure 2.

Differential dynamics of MCM loading in euchromatin and heterochromatin. (A) Projections of 3D confocal immunofluorescence images of representative cells after live-cell imaging as in Figure 1: endogenous HP1 (magenta), endogenous MCM3 (green) and DNA stained with DAPI (gray); scale bar: 5 μm. All analyses used RPE1-hTert cells expressing PCNA-mTurquoise and the DHB-mCherry CDK1/2 activity reporter. (B) Total loaded MCM3 immunofluorescence signal (y-axis) relative to CDK1/2 activity defined by the cytoplasmic versus nuclear localization of the reporter (x-axis). Cells are color coded for early (blue), middle (orange) and late (green) G1 cells defined in Figure 1. The size of data points for single cells corresponds to time since mitosis. (C) The total amount of loaded MCM3 colocalized with heterochromatin (high HP1 regions, lower purple dots) and euchromatin (low HP1 regions, higher green dots) within each cell relative to CDK1/2 activity. All five replicates are shown, n = 446. (D) Distribution of loaded MCM3 in heterochromatin (purples, lower) and euchromatin (greens, higher) in early, middle and late G1 cells as proportions of the total MCM signal per cell. Four replicates are shown in different shades; means are plotted in orange. (E) MCM3 concentration normalized to DNA/DAPI in heterochromatin (purple) and euchromatin (green) in G1 subphases. Boxplots show median (solid line) and interquartile ranges (box ends); whiskers mark the minimum or maximum. (F) MCM concentration in heterochromatin (lower purple dots) or euchromatin (upper green dots) relative to the average loaded MCM concentration in whole nuclei; mean is plotted in orange. Four replicates are shown with different shades. (G) Ratio of MCM3 concentration in heterochromatin to euchromatin; mean is plotted in orange. Four replicates are shown with different shades. For all comparisons, one-way ANOVA, Tukey post-hoc test and n (number of cells) is indicated on the figure panels or in the legend. In all panels, P-value ranges are indicated as *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001.