JCS02309 Supplementary Material

Files in this Data Supplement:

• Supplemental Table (Adobe PDF)
• Supplemental Figure 1 -

  Fig. S1. Cell cycle progression is blocked in S2 cells in response to HU and to IR. FACS profiles of DNA content after PI staining of S2 cells treated with HU (A) and FACS profiles of DNA content of cells treated with 150 Gy followed by indicated recovery times (B). After HU treatment an increase of cells in G₁ and a decrease of cells in G₂ is observed compared to control cells. The FACS data are consistent with the observed decrease in mitotic cells as demonstrated in Fig. 1. After irradiation accumulation of cells in G₂ is observed, increasing with time. Together with a decrease in mitotic cells in response to IR as presented in Fig. 1, these data indicate that S2 cells show a G₂ delay in response to irradiation. After HU or IR no increase is observed in the amount of necrotic cells, since no accumulation is observed in the amount of cells containing less than 2N DNA.

• Supplemental Figure 2 -

  Fig. S2. Grp/DChk1 expression in wild type embryos, wild type ovaries and S2 cells. Detection of Grp/DChk1 protein by western blot analysis using a Rabbit anti-Grp/DChk1 polyclonal antibody. Grp/DChk1 protein was detected in extracts from wild-type embryos (lane 1), ovaries (lane 3) and in exponentially and asynchronously growing S2 cells (lane 5). Grp/DChk1 was not detected in extracts from embryos (lane 2) and ovaries (lane 4) of homozygous grp/Dchk1 females. As a loading control, protein levels of g-tubulin were detected.

• Movie 1 -

  Movie 1. Control S2 cells progress normally through mitosis. Control S2 cells were transiently transfected with a histone H2B-GFP-expressing construct and analyzed using time-lapse confocal microscopy. In control S2 cells, condensed chromosomes aligned correctly at the metaphase plate before separation and segregation towards the spindle poles during anaphase. When cytokinesis was completed, two distinct cells were formed in which decondensed chromosomes indicated entry into G₁.

• Movie 2
• Movie 3 -

  Movies 2 and 3. Grp/DChk1-depleted cells display mitotic catastrophe in the presence of incompletely replicated and damaged DNA. Grp/DChk1-depleted S2 cells were transiently transfected with a histone H2B-GFP expressing construct, HU (Movie 2) or IR-treated (Movie 3) and analyzed using time-lapse confocal microscopy. After HU or IR treatment, Grp/DChk1-depleted S2 entered mitosis with abnormal chromosomal condensation and individual chromosome arms could not be distinguished. During mitosis, chromosomes fragmented gradually. Alignment of chromosomes in the metaphase plate was never observed in Grp/DChk1-depleted cells treated with HU and IR. Exit from mitosis was severely prolonged (~3 hours) and characterized by unequal chromosome segregation followed by the formation of micronuclei.