were mounted in Vectashield with DAPI (Vector) to stain DNA and reduce fluorescence fading. To obtain metaphase chromosome preparations, 1 ml of cell suspension was left in its well and treated for 2 hr with colchicine (final concentration 1025 M). Colchicine-treated cells were then centrifuged at 800 g for 5 min. Pelleted cells were washed in 10 ml PBS, spun down by centrifugation and resuspended in 5 ml hypotonic solution (0.5 M Na citrate) for 7 min. After further centrifugation, pelleted cells were fixed in 5 ml of methanol: acetic acid (3:1), spun down again, and resuspended in the small volume of fixative left after the removal of supernatant. 10 ml of this suspension was dropped onto a microscope slide and air-dried. All slides were mounted in Vectashield with DAPI (Vector) to stain DNA. All images were captured using a CoolSnap HQ CCD camera (Photometrics; Tucson, AZ) connected to a Zeiss Axioplan fluorescence microscope equipped with an HBO 100W mercury lamp as described previously [63]. Gray scale digital images were collected separately, converted to Photoshop format, pseudoco- lored, and merged. Phenotypic Analysis We considered 18 major phenotypic traits indicated in the headings of Figures 2 and 3, and Table S5. In addition, we observed 13 relatively rare phenotypes that are reported under the OSD (other spindle defects) and OMD (other mitotic defects) headings of Figures 2 and 3, and Table S5. Each individual phenotypic trait was considered as genuine when its frequency in a dsRNA-treated sample was significantly higher than the frequency of that trait in controls with p,0.001, using the x2 contingency test. The same trait was considered strong when its frequency was at least threefold (in the case of chromosome aberrations) or fivefold (in all the other cases) the control frequency. A phenotypic trait was considered weak when its frequency was significantly different from the control (with p,0.001), but below the above thresholds. Cells from control wells without dsRNA were systematically compared with cells treated with dsRNAs that do not elicit mitotic phenotypes. We never observed phenotypic differences between any of these cells, indicating that none of the aberrant traits we detected was due simply to the addition of random dsRNA sequences to the culture. Most of the mitotic genes identified in our screen are highly conserved and have putative human orthologs. The known function of these Drosophila genes and their human counterparts are reported in Table S6, together with the supporting references. Supporting Information Figure S1 Precocious sister chromatid separation and defective chromosome segregation after RNAi for Bub3. Ctr, control; c- meta, colchicine/hypotonic-treated metaphase chromosomes. Note in addition the elongated and bent ana/telophase spindles. Scale bar, 5 mm. Found at: doi:10.1371/journal.pgen.1000126.s001 (2.10 MB TIF) Figure S2 Lack of sister chromatid separation after RNAi for CS3 genes that encode kinetochore components. Note in addition the elongated and bent ana/telophase-like spindles in l(1)G023/ CG1558 and CG9938/hec1/Ndc80 RNAi cells. Scale bar, 5 mm. Found at: doi:10.1371/journal.pgen.1000126.s002 (4.78 MB TIF) Figure S3 Lack of sister chromatid separation after RNAi for CS3 genes that encode splicing factors. Similar to other cells in the CS3 phenocluster (Figure S2), ana/telophase-like spindles are Drosophila Mitotic Genes elongated and bent in CG3605, CG5931, and CG6015 RNAi cells. Scale bar, 5 mm. Found at: doi:10.1371/journal.pgen.1000126.s003 (4.26 MB TIF) Figure S4 Examples of mitotic figures observed after RNAi for the splicing factor gene CG10418. CG10418 knockdown results in a failure of sister chromatid separation. In some cells with ana/ telophase-like spindles, the chromosomes appear to migrate to the poles, while in others the chromosomes remain at the center of the cell. CG10418 was assigned to the CS3 phenocluster because the first type of cells is more frequent than the second one. Scale bar, 5 mm. Found at: doi:10.1371/journal.pgen.1000126.s004 (2.59 MB TIF) Figure S5 RNAi for genes of the CS3 group results in ana/ telophase-like cells that assemble irregular actin-based contractile rings despite the failure of sister chromatid separation. Note that the actin rings form in regions that contain bundled microtubules. Scale bar, 5 mm. Found at: doi:10.1371/journal.pgen.1000126.s005 (2.81 MB TIF) Figure S6 Examples of mitotic cells observed after RNAi for genes of the CS4 phenocluster. The chromosomes remain at the cell equator while the spindle elongates to assume an ana/ telophase-like morphology. The chromosomes at the center of the cell often decondense as occurs during normal telophase. Scale bar, 5 mm. Found at: doi:10.1371/journal.pgen.1000126.s006 (5.25 MB TIF) Figure S7 Defects in chromosome segregation observed after RNAi for genes of the CS5 phenocluster. Ctr, control; c-meta, colchicine/hypotonic-treated metaphase chromosomes. Note that in the c-metaphase (c-meta) from fzy RNAi cells, chromosomes are extremely condensed and the sister chromatids are separated. However, sister chromatid separation (SCS) is not observed in fzy RNAi cells with normally condensed chromosomes. We thus consider SCS as a secondary consequence of the excessive chromosome condensation, rather than a direct effect of gene product depletion. In support of this idea, RNAi for genes of the CS2 phenocluster (Figure S1) causes sister chromatid separation regardless of the degree of chromosome condensation. Scale bar, 5 mm. Found at: doi:10.1371/journal.pgen.1000126.s007 (5.70 MB TIF) Figure S8 Pole-to-pole spindle lengths (mean6SE) of ana/ telophase figures observed in the CS1-CS5 phenoclusters. The ana/telophase spindles observed in all the RNAi experiments included in the graph are significantly longer (p,0.001 in Student’s t-test) than control (ctr) spindles. Found at: doi:10.1371/journal.pgen.1000126.s008 (1.01 MB TIF) Figure S9 RNAi for genes of the CC1 phenocluster results both in a lack of sister chromatid cohesion in the heterochromatic regions of the chromosomes and in defective chromosome segregation. Ctr, control; c-meta, colchicine/hypotonic-treated metaphase chromosomes. Scale bar, 5 mm. Found at: doi:10.1371/journal.pgen.1000126.s009 (3.47 MB TIF) Figure S10 Defects in chromosome condensation and segrega- tion observed after RNAi for genes of the CC2 phenocluster. Ctr, control; c-meta, colchicine/hypotonic-treated metaphase chromo- somes. Note the extensive chromatin bridges in the ana/telophase figures. Scale bar, 5 mm. Found at: doi:10.1371/journal.pgen.1000126.s010 (3.37 MB TIF) Figure S11 Monopolar spindles, short spindles and defective chromosome segregation observed after RNAi for msps and tho2 PLoS Genetics | www.plosgenetics.org 16 July 2008 | Volume 4 | Issue 7 | e1000126