Fig. 1: Lamin A/C dynamically interacts with replication factories throughout the nucleus.

a Representative confocal microscopy image of HCT116 cells showing Lamin A/C in proximity to nascent DNA (EdU), detected by Lamin A/C:EdU PLA (magenta), Lamin A/C IF staining (yellow). While Lamin A/C is mainly detected at the nuclear periphery, its interaction with nascent DNA (EdU) is detected throughout the nucleus and in different axial perspectives (top left: XY view, bottom left: YZ view, top right: XZ view). Numerous comparable examples of this pattern were observed in two independent experiments. Scale bar, 5 µm. b Schematic representation of the Proximity Ligation assay used. EdU incorporation is followed by click chemistry with biotin-azide. Antibodies against the target protein and biotin are recognized by secondary antibodies carrying probes. When the target protein and EdU are in close proximity ( < 40 nm), probes are ligated and amplified giving rise to a fluorescent PLA signal c. Experimental design for the IF/PLA experiment in (c). The duration of the EdU pulse is adapted to allow comparable incorporation of EdU despite the genotoxic treatments. d Representative U2OS nuclei (DAPI) – untreated or treated for 1 h with 100 nM CPT or 20 nM ETP–and stained for DNA synthesis (EdU), Lamin A/C and its physical proximity to nascent DNA (Lamin A/C:EdU PLA). Scale bar, 10 μm. e Quantification of Lamin A/C PLA signals from c. Signal was quantified in at least 100 EdU+ nuclei, in 4 independent experiments. EdU- cells are used as negative control. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values +/- SD. Statistical analysis was applied on the median values, using one-way ANOVA test with Bonferroni’s post hoc correction.