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Files in this Data Supplement:
Fig. S1. PML nuclear body formation kinetics is similar in U-2 OS cells and U-2 OS cells overexpressing GFP-PML IV. Human U-2 OS cells were synchronised at the G1-S border with aphidicolin and released into S phase for 2-6 hours. PML nuclear body number versus time is shown. (Open diamonds) Curve showing the increase in PML nuclear bodies in U-2 OS cells stably expressing GFP-PML IV. (Open squares) Curve showing the increase in PML nuclear bodies in U-2 OS cells expressing endogenous levels of PML protein. Owing to the higher initial number of PML nuclear bodies in the wild-type U-2 OS cells, a curve normalized to the initial number of PML nuclear bodies in U-2 OS GFP-PML IV cells is shown (filled squares). The kinetics of the increase in PML nuclear body number through S phase is indistinguishable between the two cell types.
Fig. S2. PML nuclear body fission occurs early in S phase. U-2 OS GFP-PML IV cells were synchronized with aphidicolin and imaged after release from the synchronization step. Z-stacks were obtained every 5 minutes for 5 hours. (A) The images show the PML nuclear body distribution in a nucleus at the beginning, the middle, and the end of the time course. Note the increase in PML nuclear body number and equalisation of the relative sizes of the bodies as the cell progresses through S phase. (B) The graph shows the number of PML nuclear bodies over time as this cell enters and progresses through S phase. The increase in PML nuclear body number occurs 2-3 hours post release and plateaus at the end of the time course.
Fig. S3. PML protein levels do not increase through the cell cycle. Immunofluorescence images of PML nuclear bodies and corresponding western blot of cell extracts made from asynchronously growing GM05757 cells (A) or cells synchronised in G1, S phase and G2. PML protein levels were normalized against the intensity of the actin band (top panel). Integrated intensity of PML signal taken from fluorescence images labelled for PML were compared with the normalized PML protein levels obtained from the western analysis (top graph). The PML nuclear body number was plotted against the cells’ position in the cell cycle (bottom graph). Bar, 5 mm.
Fig. S4. PML protein exchanges more rapidly with the nucleoplasm in S phase. Immunofluorescence image of a U-2 OS GFP-PML IV cell in S phase pre-bleach. The circle indicates the nuclear body to be bleached. The smaller images to the right show the recovery of that nuclear body (top panel). FRAP curve of the PML nuclear body shown in the middle panel. The graph in the bottom panel shows the FRAP curves of PML nuclear bodies from asynchronously growing cells (interphase; n=15), and cells in S phase (n=15) and late S phase (n=30). The FRAP curves indicate that PML protein exchanges more rapidly with the nucleoplasm in S phase than in G1 phase of the cell cycle. Bar, 5 mm.
Fig. S5. PML nuclear bodies form doublets in late S phase or G2. U-2 OS cells stably transfected with GFP-PML I were labelled for PML for correlative electron spectroscopic imaging. The ESI image is segmented to show chromatin in yellow and non-chromatin based protein in blue. On the left is a PML nuclear body in an interphase cell. Note the round morphology of the PML nuclear body. On the right are two examples of PML nuclear bodies in S phase. Note the elongated morphology and doublet formation of the PML nuclear bodies.
Fig. S6. Microstructures form after actinomycin D treatment in PML–/– MEFs expressing GFP-PML IV but not GFP-PML IV-3K. PML–/– MEFs transiently expressing either GFP-PML IV or GFP-PML IV-3K were treated with actinomycin D. MEFs expressing PML IV formed microstructures after treatment with actinomycin D (white arrowheads) but not MEFs expressing PML IV-3K. Bar, 5 mm.
Fig. S7. PML nuclear body composition through S phase. (A) U-2 OS cells stably expressing GFP-PML IV were synchronized with aphidicolin at the G1-S-phase border. Cells were then released and fixed at 2 hour intervals. Cells were immunolabeled for SUMO-1 and Sp100. The merged image shows that all PML nuclear bodies in S phase contain PML, Sp100, and SUMO-1. (B) The relative levels of PML, Sp100, and SUMO-1 in PML nuclear bodies were determined by line scans between the asterixes in the merged immunofluorescence images of U-2 OS cells. At all time points assessed in S phase both parental PML nuclear bodies and microbodies contained all three proteins, indicating that their composition is similar. Bar, 5 mm.
Movie 1. PML nuclear bodies undergo multiple fission events in S phase. Human U-2 OS cells stably transfected with GFP-PML IV were synchronised at the G1-S-phase border with aphidicolin for 24 hours and observed using 4D immunofluorescence microscopy. The white arrowheads denote PML nuclear bodies fission events. Z-stacks were collected once every minute for 60 minutes. The movie is accelerated to 5 frames per second.
Movie 2. PML nuclear bodies become more dynamic and deform and elongate prior to fission. A single fission event from Supplemental Movie 1 (marked by the white arrowhead) is shown at 13 magnification. The same fission event is then shown at 33 magnification and at an angle. Note the deformation of the PML nuclear body prior to fission. Z-stacks were collected once every minute for 60 minutes. The movie is accelerated to 5 frames per second.
Movie 3. PML nuclear bodies undergo fusion in S phase. Human U-2 OS cells stably transfected with GFP-PML IV were synchronised at the G1-S-phase border with aphidicolin for 24 hours and observed using 4D immunofluorescence microscopy. A fusion event between two PML nuclear bodies is marked by the white arrowhead. Z-stacks were collected once every minute for 60 minutes. The movie is accelerated to 5 frames per second.
Movie 4. PML nuclear body dynamics in a non-S-phase cell. Human U-2 OS cells stably transfected with GFP-PML IV were observed using 4D immunofluorescence microscopy. PML nuclear bodies in a non-S-phase cell are less dynamic than PML nuclear bodies in an S-phase cell. Z-stacks were collected once every minute for 30 minutes. The movie is accelerated to 5 frames per second.
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