JCS02703 Supplementary Material

Files in this Data Supplement:

• Supplemental Table - Adobe PDF

• Supplemental Figure 1 (Adobe PDF) -

  Fig. S1. Quantification of production of ROS and death in fission yeast cells. (A) Ten thousand cells were analyzed by flow cytometry. The results are displayed as scatter plots, with increasing green fluorescence (ROS) displayed on the horizontal axis and increasing red fluorescence (PI) displayed on the vertical axis. (B) The same results were displayed in histogram form, with increasing amounts of green fluorescence (ROS) on the horizontal axis. Using FlowJo software, gates were set up as indicated by the horizontal bars. The number of cells (out of 10,000 analyzed) appearing in each of the gated regions is displayed over the horizontal bars. Because cells display autofluorescence even in the absence of dye, we measured the extent of staining in the absence as well as the presence of dyes. When the results were displayed as scatter plots of red versus green fluorescence as in (A), it was evident that the presence of dyes produced an increase in green but not red autofluorescence. In addition, a few wild-type cells and a much larger number of dfp1_D13-240 cells stained significantly more strongly in green, yellow and red. To identify and count these cells, we set up gates as illustrated in (B), which illustrates gating for green fluorescence. A similar procedure was used for red fluorescence. The gate boundaries were set up to include all cells in the stained populations having fluorescence signals higher than those in the unstained populations. In each case, 10,000 cells were scanned. In other figures, we show only the counts of cells (per 10,000) whose fluorescence exceeded the lower gate threshold (numbers over horizontal bars in B).

• Supplemental Figure 2 (Adobe PDF) -

  Fig. S2. HU does not significantly increase the already elevated ROS production and the PI staining of dfp1 N- and C-terminal deletion mutants. Incubation with or without HU and staining for ROS and PI were carried out as in Fig. 7.

• Supplemental Figure 3 (Adobe PDF) -

  Fig. S3. Repetition of the experiment of Fig. 8. In this case, addition of HU further stimulated ROS production in the cdc2-Y15F strain.