JCS03144 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Treatment of Arabidopsis protoplast with NaN₃ and 2-deoxyglucose. (A) ATP levels are given as the percentage of ATP levels of untreated protoplasts normalized to 100%. Protoplasts were assayed for ATP levels at the time points (indicated on figure) after adding NaN₃ and 2-deoxyglucose with a firefly luciferase-based ATP detection assay. Error bars are ±s.e.m. of three replications. (B) Cells were stained with Fluorescein diacetate (FDA) showing that cells remained alive during the course of experiments.

• Movie 1 -

  Movie 1. FRAP analyses of GFP-SR45 in control and fixed cells. The entire dataset of images of Fig. 1 is animated in this movie. Circles indicate the areas that were bleached.

• Movie 2 -

  Movie 2. FRAP analysis of the effect of ATP-depletion on mobility of GFP-SR45 in speckles. The entire dataset of images of Figs 2A and B was saved as full resolution images using the LSM 510 software and animated using Adobe Premiere 6.5. Circles in each panel indicate the bleached speckles.

• Movie 3 -

  Movie 3. FRAP analysis of the effect of ATP-depletion on mobility of GFP-SR45 in nucleoplasm. The entire dataset of images of Fig. 2C and D was animated using Adobe Premiere 6.5. Circles in each panel indicate the bleached nucleoplasmic area.

• Movie 4 -

  Movie 4. FLIP analyses of the ATP-depleted GFP-SR45 cells. The entire dataset of images of Fig. 2K and 2L was imported into Image ProPlus and animated. The nuclei in each panel were farther apart in the original captured images with vast blank space between them. To eliminate these spaces, these nuclei were moved closer to each other across the whole stack using Adobe Premiere 6.5. Circles in each panel indicate the repeatedly bleached area.

• Movie 5 -

  Movie 5. FRAP analyses of the effect of actinomycin-D and staurosporine on the mobility of GFP-SR45. The entire dataset of images of Fig. 5A, 5B and 5C was animated using Adobe Premiere 6.5. Circles in each panel indicate the bleached speckles. (B) Viability of protoplast after treatment with NaN₃ and 2-deoxyglucose were assayed by staining with fluorescein diacetate (FDA). Staining of ATP-depleted protoplasts shows that a majority of the protoplasts were alive. Bars, 100 μm.