JCS02817 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Enrichment of DAXX to PML nuclear bodies during the cell cycle. (A) HeLa cells were labelled for PML (green), DAXX (red), DAPI (blue), phosphorylated histone H3 (inset, pH3), and cyclin A (not shown). A combination of phosphorylated histone H3 at serine 10 and DAPI was used to identify cells at different stages of mitosis. By the beginning of mitosis, Daxx-enriched foci in the nucleus started to decrease and from prometaphase to early G1, only a faint background staining was visible. Both early G1 and G1 cells were negative for cyclin A, however the early G1 cells had a more condensed DAPI staining, less developed nucleoli, and cytoplasmic aggregations of PML. White arrowheads indicate the position of MAPPs in early G1 HeLa cells and yellow arrows indicate PML nuclear bodies enriched in Daxx above nucleoplasmic levels. At mid-G1, Daxx foci are enriched at PML nuclear bodies, however at G2 Daxx is more nucleoplasmic and less enriched at PML nuclear bodies. Bar, 5 mm.

• Supplemental Figure 2 -

  Fig. S2. Localisation of Sp100 to PML nuclear bodies and dynamics of nuclear membrane at different stages of cell cycle. HeLa cells at different stages of cell cycle were labelled for PML (yellow), Sp100 (red), Lamin A/C (green), and DAPI (blue). Throughout interphase (G1 and G2) the majority of PML nuclear bodies contain both PML and Sp100. PML nuclear bodies lose Sp100 as the NE breaks down at prophase. PML protein was associated with chromatin in anaphase and telophase, however these mitotic accumulations of PML protein (MAPPs) lack Sp100. During cytokinesis, Sp100 was seen to associate with PML in daughter nuclei, correlating with the reformation of the nuclear membrane. These data indicate that PML nuclear bodies are re-established in daughter nuclei concurrently with the reformation of the NE. White arrowheads indicate the position of MAPPs in early G1 HeLa cells and yellow arrows indicate PML nuclear bodies (or MAPPs) enriched in Sp100 above nucleoplasmic levels. Bar, 5 mm.

• Supplemental Figure 3 -

  Fig. S3. Comparison of the localisation of MAPPs and MIGs in mitosis and early G1. (A) Asynchronous HeLa cells were fixed, labeled for PML (green), SC35 (red) and DNA was counterstained with DAPI (blue). Cells at various stages of mitosis were analyzed and data indicated that MAPPs did not localize with MIGs. (B) HeLa cells were prepared by mitotic shake-off from asynchronous cultures and plated on coverslips and imaged after 1-2 hours. Cells were fixed and processed for the immunofluorescence detection of PML (green) and SC35 (red). Two daughter cells in early G1 are shown. Although MAPPs were seen in the cytoplasm of G1 cells several hours after the completion of mitosis, all SC35-containing MIGs had disappeared by cytokinesis. These data indicated that the loss of MIGs and MAPPs from the cytoplasm of mitotic/G1 cells (respectively) is temporally distinct and therefore unlikely to be mechanistically coupled. Bar, 5 mm.

• Movie 1 -

  Movie 1. Dynamics of PML nuclear bodies in U20S cells during mitosis. Unsynchronised U20S cells expressing GFP-PML IV (a gift from J. Taylor, Medical College of Wisconsin) were grown on glass coverslips and placed in a live-cell environmental chamber (constructed in our laboratory) containing CO2-equilibrated growth medium (JMEM, 10% FBS) and cells were maintained at 37°C while being imaged for using a Leica Microsystems DMRA2 upright microscope. DIC and fluorescence images were taken every 4 minutes for approximately 10 hours. The resulting images were collated, cropped, pseudo-colored and compiled into an AVI using ImageJ software (NIH). The movie plays at 5 frames/second with each frame representing 4 minutes. Two cells in the centre of the field of view pass from G2, through mitosis and into G1. GFP-PML nuclear bodies in yellow can be seen to increase in mobility as cells enter mitosis, at which time they begin to fuse into larger mitotic accumulations of PML protein (MAPPs).

• Movie 2 -

  Movie 2. Aggregation of MAPPs during mitosis in U20S cells expressing GFP-PML IV. Unsynchronised U20S cells expressing GFP-PML IV were imaged in mitosis as in Movie S1. DIC and fluorescence images were taken to identify mitotic cells and a cell in metaphase was imaged every 20 seconds for approximately 3 minutes. The resulting images were collated, cropped, and compiled into an AVI using ImageJ software (NIH). The movie plays at 1 frames/second with each frame representing 20 seconds. Several MAPPs are shown to aggregate together over the time imaged.

• Movie 3 -

  Movie 3. Comparative FRAP analysis of MAPPs in mitosis and PML nuclear bodies in interphase. Unsynchronised U20S cells expressing GFP-PML IV were imaged in mitosis as in Movie S1 and a mitotic cell and an interphase cell were chosen for fluorescence recovery after photo-bleaching (FRAP) analysis as described in the Methods and Materials. A single GFP-PML IV-containing MAPP in the mitotic cell (left panel) or PML nuclear body in one of two adjacent interphase cells (left-hand cell in the right panel) were chosen for FRAP analysis. Images were taken before and after photo-bleaching at intervals of 1 minute for 11 minutes in total. The resulting images were collated, cropped, and compiled into an AVI using ImageJ software (NIH). The movie plays at 1 frame/second with each frame representing 1 minute. A white circle indicates the position of the photo-bleached MAPP or PML nuclear body. The entire mitotic cell was imaged (left panel) to allow the location of the bleached MAPP to be tracked, whereas only the ROI containing the interphase PML nuclear body was imaged and superimposed on the pre-bleached image of the interphase cell (right panel). The photo-bleached MAPP fails to recovery over the time period imaged whereas the interphase PML nuclear body recovers fully within 9 minutes post bleach.