Supplementary Figure S1 a, Schematic diagrams of the MAD2L1 and Cyclin A (CCNA) promoter regions. Black dots represent predicted E2F binding sites (see part b). Shown below are the primer sequences used in the study. b, Nucleotide sequence and location of the predicted E2F binding sites relative to the transcription start site (TSS) in respective promoters. mRNA sequences of human Mad2 (NM_002358) were retrieved from NCBI nucleotide database and analyzed for E2F binding sites and contextual motifs as described15. Chromatin immunoprecipitation in IMR90 cells was carried out as previously described 16 using an E2F-1 specific antibody (sc-193, Santa Cruz). For reporter assays, genomic fragments corresponding to the Mad2 promoter were amplified by PCR and cloned into the pGL3-Basic luciferase reporter vector (Promega). U-2OS and IMR90 cells were transfected using FuGene 6 (Roche) as conducted in ref. 15. Supplementary Figure S2 a, Immunohistochemical detection of Mad2 levels in highly proliferating organs (thymus and tonsil). Ki-67 staining is shown as a proliferation marker. b, Photomicrographies of transitional cell carcinomas of the bladder showing Mad2 and Ki-67 expression. Original magnifications: x400. Supplementary Figure S3 a, Mad2 immunofluorescence staining (FITC) of Mad2-transduced IMR90 fibroblasts. Phase-contrast facilitates the identification of multinucleated cells. PI was used to counterstain the nuclei. b, -tubulin staining (FITC) in IMR90 cells transduced with Mad2 retroviruses, as in vectorinfected cells. Nuclei stained with DAPI. Supplementary Figure S4 a, Analysis of mitosis progression, considering first stage: from prometaphase to late anaphase (chromosome segregation and beginning of cell division), and second stage: from late anaphase to completed cytokinesis and reattachment of daughter cells. These two periods were defined based on phase-contrast videomicroscopy. The differences between vector and the rest were statistically significant (Student t-test; GraphPad Prism 3.0cx program for Macintosh). Western Blot analysis of cyclin B1 (cycB) (b) and securin/Pds1 (c) protein levels in shRb- (R), Mad2- (M), and vector controltransduced HCT116 and IMR90, respectively. Total cellular protein was extracted from cells synchronized by double thymidine blockade in G1/S and released into nocodazole. Histone H3-P levels indicating mitotic progression and APC2 levels are shown. -tubulin serves as loading control. d, Representative Western blot analysis showing E1A and E2F protein levels in HCT116 cells ectopically expressing E2F, E1A or a vector control together with either a Mad2 hairpin (shMad2) or a hairpin control (sh-con). Supplementary Videos. Time lapse videomicroscopy images of dividing NIH3T3 cells expressing Histone-GFP. Normal cell division in vector-transduced NIH-3T3 cells by phase contrast (Video A) and green fluorescence (Video B). Representative examples of abnormal cell division with prolonged or failed cytokinesis in cells transduced with shRb (Video C, phase contrast, and D, GFP) or Mad2 (Videos E and F, phase contrast). Illustrative examples of problems in chromosome segregation, such as lagging or eventually lost DNA material, or asymmetrical excision, in E1A- (Video G, GFP) or Mad2-expressing NIH-3T3 cells (Video H, GFP). Similar phenotypes were observed in shRb- (video I, GFP) or Mad2- (video J, GFP) transduced HCT116 cells. Cells were transfected with pBOS-H2B-GFP construct (Pharmingen, BD) 24h before initiating the analysis. Real time and in situ microscopy used METAMORPH software on a Zeiss Axiovert 200M microscope and a Roper Scientific Micromax 1300YHS camera27. (QuickTime, A: 8.939 MB; B: 273 KB; C: 8 MB; D: 6.545 MB; E: 7.565 MB; F: 5.387 MB; G: 382 KB; H: 809 KB; video I: 632 KB, J: 973 KB).