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).