JCS03068 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Co-localization between ssDNA and PML. The indicated cell lines were metabolically labeled with BrdU for 24 hours. They were then irradiated with UV (10 J/m²) and subsequently incubated in the presence of 4 mM caffeine for 2 hours. Following fixation and immunofluorescence labeling of cells, images representing single confocal sections through the nuclei were generated using a laser confocal scanning microscope. Right panels show fluorescence intensity histograms of BrdU (red) and PML (green) labeling through the red line shown in the left panels. White stippled lines indicate the nuclear boundaries.

• Supplemental Figure 2 -

  Fig. S2. Effect of ATR and ATM depletion on ssDNA focus formation. (A) Western blot of extracts prepared from GM-847 cells at 72 hours after transfection with the control siRNA (GL2) and siRNAs specific for ATM and/or ATR. β-actin was used as a loading control. (B) GM-847 cells seeded on coverslips were transfected with the indicated siRNAs. On day 2 following transfection, BrdU was added to the cell culture medium. 24 hours later, cells were treated with 10 J/m² UV light and subsequently cultured for 1 (T=1h) and 2 (T=2h) hours before fixation and analysis by immunofluorescence labeling. Data represent the percentage of cells containing detectable ssDNA foci within PML-NBs. For each sample more than 500 cells were evaluated. The average of two independent experiments ± s.d. is shown. (C) GM-847 cells were treated with siRNAs and UV-irradiated as described in B. Subsequent to irradiation, cells were grown in the presence or absence of 4 mM caffeine for 2 hours prior to fixation and immunofluorescence labeling. Data represent the percentage of cells containing detectable ssDNA foci within PML-NBs. For each sample more than 500 cells were evaluated. The average of two independent experiments ± s.d. is shown.

• Supplemental Figure 3 -

  Fig. S3. Effect of PML-depletion on UV-induced ssDNA focus formation in GM-637 cells. (A) Western blot of nuclear matrix extracts prepared from cells at 48 hours following transfection with PML or GL2 (control)-specific siRNAs. The β-actin protein is used as a loading control. (B) Cells were transfected with the indicated siRNAs on day 1, and on day 2 the growth medium was supplemented with 10 μM BrdU. On day 3 (24 hours following addition of BrdU) cells were UV-irradiated and subsequently grown in the presence or absence of 4 mM caffeine for 2 hours before fixation. For each sample the presence or absence of ssDNA foci was determined in more than 400 cells. Data represent the average of 2 independent experiments ± s.d.

• Supplemental Figure 4 -

  Fig. S4. Effect of etoposide and doxorubicin on ssDNA focus formation and cell cycle progression in GaMg cells. Cells were transfected with control siRNAs (GL2) or PML-specific siRNAs, and at 24 hours following transfection the culture medium was supplemented with 10 μM BrdU. At 48 hours post-transfection the BrdU-containing medium was removed and cells were incubated for another 24 hours in medium containing the indicated concentrations of etoposide or doxorubicin. (A) Quantitative determination of cells containing ssDNA foci within PML-NBs. For each sample more than 500 immunofluorescence-labeled cells were evaluated. Bars represent the average of two independent experiments ± s.d. (B) Analysis of To-Pro-3-stained cells by laser scanning flow cytometry.

• Supplemental Figure 5 -

  Fig. S5. Effect of PML-depletion on UV-induced apoptosis in GM-637 cells. Cells were transfected with PML-specific siRNAs or control siRNAs (GL2) and subjected to 10 J/m² UV-irradiation or non-irradiated on day 3 following transfection. Cells were subsequently incubated in the presence or absence of 4 mM caffeine for 2 hours prior to analysis. (A) Cells were fixed, stained with Hoechst and evaluated by immunofluorescence microscopy for the presence or absence of condensed chromatin. For each sample, more than 500 cells were evaluated. The data represents the mean ± s.d. of two independent experiments. (B) Analysis of apoptotic DNA fragmentation by pulse field gel electrophoresis. The 50-300 kb apoptotic DNA fragments are indicated.

• Movie 1 -

  Movie 1. GM-847 cells expressing YFP-PML. The movie shows the movement of PML-NBs within a single cell nucleus during a 90 minute period immediately following UV-irradiation and addition of growth medium containing 4 mM caffeine. Images were captured every 30 seconds.