A) The images show γH2Av (white in top panel and red in bottom panel) and C(3)G (green) IF in whole-mount germaria from wild type, Su(var)3-9 and mei-W68; Su(var)3-9. C(3)G is part of the synaptonemal complex and used to distinguish oocytes from nurse cells, both of which contain DSBs. Each image is an optical section; bar = 7 mm. B) and C) The graphs show the average numbers and volumes (relative to total nuclear volumes) of γH2Av foci in nurse cells and oocytes from wild type, Su(var)3-9 and mei-W68; Su(var)3-9. Both quantitation methods showed that γH2Av foci in Su(var)3-9 nurse cells were significantly increased over wild type (p<0.001). γH2Av foci in Su(var)3-9 oocytes were significantly increased over wild type (p<0.001). The numbers of γH2Av foci in mei-W68; Su(var)3-9 nurse cells and oocytes were lower than Su(var)3-9 alone and not significantly different from wild type (p<0.001). Error bars indicate standard deviations, p values were calculated by Student's t test, and n>15 for each cell type. D) Combined γH2Av IF (red) and satellite FISH (green) in wild-type and Su(var)3-9 germaria; C(3)G (grey) staining identifies the oocytes. Percent of oocyte and nurse cells that displayed overlap between γH2Av and satellite signals are shown. Each image is an optical section, and cells are 5 mm wide.

A) Analysis of developmental progression for wild type and Su(var)3-9 animals. Animals in the two groups laid comparable numbers of eggs. In all three assays, the p values comparing Su(var)3-9 to wild type are <0.001 by Student's t test. n>150 for each genotype. B) The graph shows lifespan analysis of wild type and Su(var)3-9 adult female flies. Su(var)3-9 females displayed significantly shorter lifespan than wild type (p<0.01 by Wilcoxon signed rank test). n>50 female flies.

A) The chart lists the viability of the double mutants of Su(var)3-9 with mutations in the DNA damage checkpoint and mitotic checkpoint pathway. Viability was calculated relative to single homozygous checkpoint mutants, which are less viable than Su(var)3-9 single mutants. Progeny counts are in Table S1. P values were calculated by the Chi-square test. B) Cell cycle analysis of wild type, Su(var)3-9, grp; Su(var)3-9 and lok; Su(var)3-9 mutant imaginal discs and brains. The percentages of G1 cells in the two double mutants were higher than wild type and single Su(var)3-9 mutants. The percentages of S phase cells in the two double mutants are lower than wild type, but do not differ from Su(var)3-9. The percentages of G2 cells in the double mutants are lower than wild type and Su(var)3-9. The mitotic indices in the double mutants were lower than Su(var)3-9 and do not significantly differ from the wild type. P<0.05 for all tests that show significant differences between wild type and Su(var)3-9; p values were calculated by Student's t test, and n>1000 cells for each genotype.

A) γH2Av (red) and Rad51 (green) IF in whole-mount diploid tissues from wild type and Su(var)3-9 mutants are shown. Each image is an optical section. γH2Av- and Rad51-positive cells in Su(var)3-9 are 96- and 11- fold increase over wild type. The p values were <0.01 by the Student's t test, and n>800 cells for each group. The scale bars = 25 mm in γH2Av IF images and 8 um in Rad51 images. B) An optical section shows a wild type diploid cell stained with DAPI; dashed lines encircle the DAPI-bright regions. Bar = 0.8 mm. Right panel are optical sections of Su(var)3-9 mutant diploid cells stained by the TUNEL assay (red foci). The foci are double-stranded breaks recognized by TdT. Enlarged images showed examples of TUNEL foci in DAPI-weak and DAPI-bright regions. Bar = 4 mm. C) shows quantitative analysis of γH2Av and TUNEL signal localizations in wild type and Su(var)3-9 cells. The distribution of γH2Av and TUNEL signals in DAPI-weak regions do not differ significantly between wild type and Su(var)3-9 (p>0.05 by Chi-square test; n>40 for each genotype). Compared to wild type, γH2Av foci localized to DAPI-bright regions in Su(var)3-9 is 96-fold higher and 88-fold for TUNEL (p<0.001 by Chi-square test; n>35 for each genotype).

A) DAPI staining of mitotic chromosomes from wild type and Su(var)3-9 mutant diploid cells. Structural defects in Su(var)3-9 mitotic chromosomes are indicated by white arrows. Each image is an optical section; bar = 2 mm. B) The chart shows quantitation of defective Su(var)3-9 mitotic chromosomes. Some mitotic chromosomes exhibited more than one defect. C) Chromosome painting of mitotic chromosomes from wild type and Su(var)3-9 mutant. Red = 3rd chromosomes, green = 2^nd chromosomes, and blue = X chromosomes. Fourth and Y chromosomes are only stained with DAPI. Structural defects, such as deletions and translocations, are indicated by white arrows. Each image is an optical section; bar = 2 mm. D) Quantitation showed that 1.1% of the Su(var)3-9 mitotic chromosomes exhibited structural defects, compared to 0% for wild type (p<0.05 by Chi-square test). E) The diagram illustrates the genetic assay used to quantitate genome instability (loss of heterozygosity) in wild type and Su(var)3-9 animals. Wild type virgin flies were mated with males hemizygous for w⁻ (recessive white¹¹¹⁸ mutation) to produce females heterozygous for w⁻ and w⁺. A clone of w− adult eye cells (pictured) arises during larval development when the w⁺ allele is lost due to mitotic recombination, deletion, or chromosome loss events. f) Quantitative analysis of w− clone frequencies in wild type and Su(var)3-9 animals. The p values were calculated using the Chi-square test.

A) The histograms show cell cycle stage analysis of wild type and Su(var)3-9 cells. The percent of G1 cells do not differ significantly (p>0.05). The percentage of S phase cells in Su(var)3-9 is significantly lower than wild type (p<0.05). The percent of wild-type cells in G2 is significantly lower than in Su(var)3-9. Mitotic indicex in Su(var)3-9 is 4-fold over wild type (p<0.001). The percent of apoptotic cells (whole nuclei contain TUNEL signals, instead of foci) in Su(var)3-9 is 9-fold over wild type (p<0.001). P values were calculated by Student's t test, and n>1000 cells for each genotype. B) The chart shows γH2Av foci numbers in G1, S, and G2 cells of wild type and Su(var)3-9. Analysis of the ratios of γH2Av foci to total cell numbers during the cell cycle in wild type and Su(var)3-9. γH2Av foci in Su(var)3-9 cells increased over wild type in G1, S, and G2 (p<0.01). P values were calculated by Chi-square test, and n>40 cells for each genotype.

A) Analysis of the ratios of γH2Av foci to total cell numbers at different cell cycle stages are shown for wild type and dcr-2. γH2Av foci in dcr-2 cells only increased during S phase (p<0.05). P values were calculated by Chi-square test. B) The histograms show cell cycle stage analysis of wild type and dcr-2 cells. The percent of G1 cells in the two groups do not differ significantly (p>0.05). The percentage of S phase cells is not significantly lower in dcr-2 compared to wild type (p>0.05), but the percent of wild-type cells in G2 is significantly lower than in dcr-2 (p<0.05). The mitotic index in dcr-2 cells is 11-fold over wild type (p<0.001), and the percent of apoptotic cells (whole nuclei contain TUNEL signals, instead of foci) in dcr-2 is 18-fold over wild type (p<0.001). P values were calculated by Student's t test, and n>1000 cells for each genotype. C) The chart lists the viability of double mutants of dcr-2 with mutations in the DNA damage checkpoint and mitotic checkpoint pathways. Viability was calculated relative to single homozygous checkpoint mutants, which are less viable than dcr-2 single mutants. Progeny counts are in Table S1. P values were calculated by the Chi-square test.