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Fig. S1. Immunoelectron microscopy of telophase cells expressing GFP-BAF. HeLa cells stably expressing GFP-BAF were fixed with 4% formaldehyde in PBS for 2 hours at room temperature, then washed with PBS three times for 5 minutes each. At this point, fluorescence image data were obtained by the DeltaVision microscope system using a PlanApo 60× oil objective lens as described in Materials and Methods. Then, cells were permeabilized for 30 minutes at room temperature with 0.25% saponin dissolved in PBS containing 5% bovine serum albumin, washed with PBS three times for 5 minutes each, and treated for 30 minutes with a blocking solution of PBS containing 0.005% saponin, 10% bovine serum albumin, 10% normal goat serum and 0.1% cold-water fish skin gelatin. Then, cells were treated with anti-GFP-antibody (No. 17 rabbit polyclonal antibody, a gift from Nobuhiro Nakamura, Kanazawa University, Kanazawa, Japan) in the blocking solution overnight. After washing with PBS containing 0.005% saponin six times for 10 minutes each, cells were incubated with goat anti-rabbit IgG conjugated with nanogold (Nanoprobes, Yaphank, NY) in the blocking solution for 2 hours, and then washed with PBS containing 0.005% saponin six times for 10 minutes each, and with PBS twice for 5 minutes each. The cells were fixed again with 1% glutaraldehyde in PBS for 10 minutes, and washed with 50 mM HEPES (pH 5.8) three times for 3 minutes each. After washing with distilled water once for 1 minute, cells were reacted with 2012 silver-enhancement kit solution (Nanoprobes) in the dark for 6 minutes at 20°C, and washed with distilled water in the dark three times for 1 minutes each, and postfixed with 0.5% OsO4 in phosphate buffer (pH 7.4) for 90 minutes at 4°C, and washed with distilled water three times for 3 minutes each. After that, cells were embedded in Epoxy resin and imaged as described in Materials and Methods. (A) An immuno EM image. Dark dots are signals for GFP-BAF. (B) Drawings superimposed on the EM images indicating GFP-BAF signal-enriched regions (green), the NE (red), and microtubules (yellow). Scale bar: 500 nm.
Fig. S2. EM analysis of telophase cells. HeLa cells stably expressing GFP-BAF were fixed at approximately 4 minutes (A and B), or 7 minutes (C and D) after the metaphase-anaphase transition and observed by EM as described in the Materials and Methods. Right panels are a magnified view of the region marked by the red boxes in the left panels. Scale bar: 500 nm.
Fig. S3. EM analysis of telophase HeLa cells with no exogenous GFP-BAF. This analysis is used as a control for the electron dense structure observed in cells expressing GFP-BAF. HeLa cells were fixed and observed by EM as described in Materials and Methods. (A) An EM image of a whole cell. (B) A magnified image of the chromosomal region marked by the red box in A. (C) Magnified EM images of the regions (a and b) marked by a blue box in B. In the lower panels, drawings are superimposed on the EM images indicating the electron-dense region (green), the NE (red hatch), and microtubules (orange line). Scale bars: 2 µm (A), 1 µm (B) and 500 nm (C).
Fig. S4. Live-correlated EM analyses of HeLa cells expressing GFP-BAF after siRNA treatment (C,D) or luciferase siRNA treatment as a control (A,B). Magenta represents chromosomes (Hoechst 33342) and green represents GFP-BAF. (A,C) Time-lapse images of living HeLa cells stably expressing GFP-BAF were obtained every 1 minute. (B,D) A series of deconvolved 3D images of the cell after fixation: from the bottom of the cell to the top of the cell. The fluorescence images marked by a yellow box in B and D are same as shown in Fig. 8C and Fig. 8H, respectively.
Fig. S5. Indirect immunofluorescence staining of HeLa cells treated with luciferase RNAi as a control (top two panels) and with BAF RNAi (bottom tow). HeLa cells were fixed with trichloroacetic acid and stained with affinity-purified anti-BAF antibody and anti-tubulin antibody as described previously (Haraguchi et al., 2001; Haraguchi et al., 2004). A series of three dimensional image data were obtained by a DeltaVision fluorescence microscope system using PlanApo 60× oil immersion objective lens (Olympus) and deconvolved with the SoftWorx software equipped with the DeltaVision. An image of a single focal plane is shown for each cell. Color represents chromosomes in blue, BAF in green, and tubulin in red. Scale bar: 10 µm.
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