JCS031922 Supplementary Material

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• Supplemental Table S1 -

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• Supplemental Figure S1 -

  Fig. S1. Expression of PML proteins. (A) GFP-PML fusion proteins are expressed as full-length proteins. Western-blot strips of whole-cell lysates of untransfected (mock) and GFP-PML transfected U-2 OS cells were analyzed for fusion protein expression using an anti-GFP antibody. Numbers on the left refer to the position of molecular mass standards. An unspecific background band at 55 kDa (asterisk) was sometimes observed, which was also present in control lysates (see mock lane). (B) U-2 OS cells express the same pattern of endogenous PML isoforms as primary human cells. U-2 OS cells were cultured in the absence (−) or presence (+) of interferon γ (IFNγ) for 24 hours. Whole-cell extracts of these cells were then probed by western blotting to detect endogenous PML proteins using rabbit-anti-PML(209-227) antiserum. Anti-tubulin reactivity was also analyzed as a loading control. Numbers on the right refer to the position of molecular mass standards.

• Supplemental Figure S2 -

  Fig. S2. Expression of PML nuclear body components. (A) U-2 OS cells expressing the indicated GFP-PML isoforms were analyzed by indirect immunofluorescence and confocal microscopy. Images show mid-nucleus confocal sections of ToPro-3 staining (DNA), GFP signals (GFP), and anti-Sp100 immunoreactivity (Sp100). Colocalizing regions between GFP and anti-Sp100 fluorescence are also shown (Coloc.). Scale bars, 10 µm. (B) U-2 OS cells expressing the indicated GFP-PML fusion proteins were analyzed by indirect immunofluorescence and confocal microscopy. Images show mid-nucleus confocal sections of ToPro-3 staining (DNA), GFP signals (GFP), and anti-PML or anti-Sp100 immunoreactivity (Endog.). Colocalizing regions between GFP and PML or Sp100 fluorescence is also shown (Coloc.). Scale bars, 10 µm. (Right panels) Western blot strips of whole-cell lysates of U-2 OS cells transfected with the respective GFP-PML fusion constructs. Numbers on the right indicate the position of molecular mass standards. (C) U-2 OS cells expressing the indicated GFP-Sp100 fusion variants were analyzed by indirect immunofluorescence and confocal microscopy. Images show mid-nucleus confocal sections of ToPro-3 staining (DNA), GFP signals (GFP), and anti-PML immunoreactivity (Endog.). Colocalizing regions between GFP and PML fluorescence is also shown (Coloc.). Scale bars, 10 µm. (Right panels) Western blot strips of whole-cell lysates of U-2 OS cells transfected with the GFP-Sp100 variants. Numbers on the right indicate the position of molecular mass standards. (D) U-2 OS cells expressing the indicated GFP fusion proteins were analyzed by indirect immunofluorescence and confocal microscopy. Images show mid-nucleus confocal sections of ToPro-3 staining (DNA), GFP signals (GFP), and anti-PML or anti-Sp100 immunoreactivity (Endog.). Colocalizing regions between GFP and PML or Sp100 fluorescence is also shown (Coloc.). Scale bars, 10 µm. (Right panels) Western blot strips of whole-cell lysates of U-2 OS cells expressing the GFP-fusion proteins. Numbers on the right indicate the position of molecular mass standards. Due to extremely low transfection efficiencies with the PFG-BLM construct, we were not able to perform a western blot to detect this fusion protein.

• Supplemental Figure S3 -

  Fig. S3. Fitting of FRAP data using one and two component binding-diffusion models. The mean values of FRAP curves for the indicated GFP-tagged proteins (blue dots) were fitted using the diffusion-binding model (with two components at NBs) depicted in figure 3C (solid red lines), or assuming only one population (1-component model) of exchanging molecules (solid green lines). The 1-component model provided a fit only for PML I (A). However, this fit curve deviates from the measured FRAP values considerably during the first two minutes (data not shown), whereas the 2-component model fitted perfectly at all times.

• Supplemental Figure S4 -

  Fig. S4. Effect of ATP depletion on PML mobility. (A,B) Nucleoplasmic dynamics of PML in ATP depleted cells. (A) FRAP experiment on a GFP−PML-I-expressing cell after 1 hour of ATP depletion. Images show the range-indicator signals of one confocal section each. The detector gain for image aquisition was adjusted to capture the nucleoplasmic signals of the GFP fusion protein within the quantifiable pixel brightness range (0-255 in a 8-bit image, white signals). The red color, indicating PML NBs, represents oversaturated pixel brightness (>255). The boxed area within the nucleus shows the bleached region, which is also shown enlarged as insets of each image. FRAP was measured within the area depicted by the green circle to monitor GFP−PML-I dynamics outside the adjacent nuclear body. (B) FRAP of GFP−PML-I in the nucleoplasm was measured as shown in (A) and quantified. The fast and almost complete recovery indicates high mobility of GFP−PML-I through the nucleoplasm of ATP-depleted cells. (C,D) PML exchange at nuclear bodies is restored after recovery from ATP depletion. (C) FRAP experiments were performed on the same GFP−PML-I expressing U-2 OS cells during ATP-depletion (Δ ATP) or 1 hr after drug removal (washout). Images represent maximum intensity projections from 3D time-lapse FRAP data sets. Bleached regions containing a PML NB are indicated by an arrow and these are also shown as enlarged insets. (D) FRAP experiments were performed as descibed in (A) and fluorescence recovery after 10 minutes of the bleach pulse was quantified from fifteen PML NBs (three NBs × five cells) and displayed as the mean ± s.d. Scale bars, 5 µm.