Figure 1. Time-dependent differences in structural forms of PML nucleolar associations induced by doxorubicin. As schematically depicted (A), RPE-1hTERT were treated with 0.75 μM doxorubicin and diverse types of PNAs were quantified by analysis of microscopic images in several time-points as indicated in the scheme. (B) Representative images of structural categories of PNAs obtained by wide-field indirect immunofluorescence microscopy of nuclei immunostained for PML (green). Nuclear and nucleolar compartments were visualized with DAPI (blue) and TOTO-3 (red), respectively. The images were captured with 63×/1.4 objective. Bar, 10 μm. (C) The percentage of cells containing specific structural subtypes of PNAs categorized as 'circles', 'caps', 'forks' and 'PML nucleoli-derived structures' (PML-NDS) was estimated. Over 200 cells in three biological replicates were evaluated for each time-point. Results are presented as a mean ± s.d. (D) Indirect immunofluorescence showing the colocalization of PNAs with proteins of PML nuclear bodies (PML-NBs). PML (green) and PML-NBs proteins (red) are visualized with respective antibodies, the nucleus was stained with DAPI (blue) and the nucleolus with TOTO-3 (cyan). The images were captured with 63×/1.4 objective. Bar, 10 μm.