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Fig. 2. Localisation of PML-containing structures in human cells during mitosis and early G1 by light and electron microscopy. (A,B) Immunolocalisation of mitotic accumulations of PML protein (MAPPs) (red) in a mitotic HeLa cell grown on a coverslip (A) or a HeLa mitotic spread prepared by cytospin (B). White arrowheads indicate examples of MAPPs closely associated with chromosomes. DNA is counterstained with DAPI (blue). Note that the image shown in panel B is of two overlapping mitotic spreads. (C-F) Correlative light and electron microscopy of MAPPs associated with mitotic chromosomes. Mitotic HeLa cells were shaken from flasks and plated on coverslips before processing for correlative light and ESI. (C) A correlative fluorescence image of an ultra thin section (70 nm) of a mitotic SK-N-SH cell grown on a coverslip is shown labelled for PML (yellow) and phospho-H3 (blue). This image was used to correlate the location of MAPPs (yellow) in the electron micrograph in D. (D) Low magnification electron micrograph (155 keV phosphorus-enriched) of the mitotic cell in C. (E) Composite electron micrograph of nitrogen and phosphorus maps collected by ESI, which details the protein-based structures (blue) and chromatin (yellow) of the mitotic cell. The white arrowhead indicates the MAPP seen in B. A white box indicates the area of interest containing a MAPP to be imaged at higher magnification. (F) Higher magnification composite ESI micrograph of the MAPP within the white box indicated in panel E. Small white arrows indicate positions where the MAPP (blue structure, white arrowhead) makes direct contact with chromatin (yellow). (G,H) PML nuclear bodies form in early G1 nuclei by entrapment of MAPPs and/or nuclear import of PML protein. Asynchronous HeLa cells were fixed and processed for the immunogold detection of PML by ESI. (G) An ESI micrograph of a HeLa cell in late cytokinesis is shown. In this micrograph a PML accumulation appears to have been entrapped by the reforming nuclear envelope, as indicated by a cluster of immunogold labelling for the PML protein (white dots). A region of interest containing this nascent PML nuclear body (defined by the white square), is shown as an inset. The arrowheads indicate nuclear pore, though the region to the left of the arrowheads, close to the MAPP shows a region where the nuclear envelope has not completely re-formed. (H) An ESI micrograph of a HeLa cell in early G1 is shown in which two PML nuclear bodies can be seen, one near the nuclear envelope (defined by the white box; shown at higher magnification in the inset) and a smaller PML nuclear body in the lower left corner of the micrograph (white asterisk). Two nucleopores are shown associated with immunogold complexes directed against PML (inset). Bars, 10 µm (A,B); 5 µm (C,D); 1 µm (E,F); 0.5 µm (G,H).
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