beyond the adenoma stage, suggesting a potential connection between modifiers of ploidy and disease progression. Our observations are consistent with the models that posit the evolution of tumor cells (Fig. 7) (Nowell, 1976; Merlo et al., 2006). In this scenario, tetraploid cells in the small intestine form a pool of cells from which aneuploid and/or neoplastic cells sample a variety of genotypes, most of which will fail to give rise to a via- ble cell and, therefore, avoid the cancer phenotype. These observa- tions argue for a direct path from mitotic defects to cancer and raise important questions as to the molecular and cellular state of these cells at this early stage. It is abundantly clear from our analyses that aneuploidy is a constant and early feature of intestinal epithelium expressing clinically relevant mutations in APC. This is the first evidence that a mutation that predisposes patients to colorectal cancer induces chromosomal aneuploidy before any other changes associated with cancer, therefore placing genomic instability in a position to play a causative role in tumor progression. Materials and methods Cell lines and antibodies Regions encoding the indicated fragments of human APC and a 13-myc epitope tag were subcloned for expression, and stable human 293 cell lines were generated as previously described (Green and Kaplan, 2003). Cells were transfected with EB1-GFP (pIK131; provided by J. Tirnauer, University of Connecticut Health Center, Farmington, CT), enhanced GFP– tubulin (pFM258; provided by F. McNally, University of California, Davis, Davis, CA), or enhanced YFP (EYFP)–histone 2B (provided by G. Kops, University Medical Center, Utrecht, Netherlands). Antibodies against c-myc (9E10; Santa Cruz Biotechnology, Inc.), tubulin (Tub2.1 or FITC-conjugated Tub2.1; Sigma-Aldrich), human centromere antigens (anticentromere anti- body; Antibodies, Inc.), γ-tubulin (GTU-88; Abcam), or β-catenin (BD Bio- sciences) were used according to the manufacturers’ recommendations. Antibodies against Numa were used at a 1:200 dilution for immunohisto- chemistry. The c-APC antibody was made by injecting rabbits with a puri- ﬁed recombinant fragment of APC encoding amino acids 2,038–2,843 of the human protein. Speciﬁcity of the serum was conﬁrmed by immuno- blotting and on APC mutant human tumor cells. Cell culture Cells were cultured in DME high glucose medium (Cellgro) supplemented with 10% FCS, 2 mg/ml L-glutamine, 1 mM sodium pyruvate, 200 μg/ml penicillin, and 50 U/ml streptomycin. Cells were maintained at 37°C and 5% CO2. Plasmid expression vectors for the indicated fusion proteins were transfected into the indicated cell lines using the FuGene 6 reagent (Roche). Cells were treated with ponasterone (Invitrogen) to induce expression of the APC allele 24 h after transfection and were maintained in ponasterone- containing media for 24–48 h before analysis. In the long-term expression experiments, the media was changed every 3–4 d, and fresh ponasterone was added to the media. Microscopy Cells were grown on coverslips and processed for immunoﬂuorescence microscopy as previously described (Green and Kaplan, 2003). Fluores- cence was collected using an inverted microscope (IX70; Olympus) ﬁtted to an imaging system (DeltaVision RT; Applied Precision). Images were re- corded using a 60× 1.4 oil immersion lens (for ﬁxed images, live cell imaging, and intestinal sections; Olympus) or a 20× 0.75 oil immer- sion lens (for intestinal sections; Olympus) and a camera (CoolSnap HQ; Roper Scientiﬁc). For live cell imaging, cells were grown on 50-mm round coverslips before being transiently transfected with the indicated expres- sion constructs, were allowed to recover for 24 h, and then were induced with ponasterone (as indicated) for 24 h. The coverslip was ﬁtted to a live cell imaging chamber (FCS2; Bioptechs) and maintained at 37°C on the microscope stage. The pH of the media during ﬁlming was buffered by the addition of 10 mM Hepes, pH 7.4. An open shutter system was used to rapidly collect 2–4 μm of data at each time point of the video. Deconvolu- tion and image analysis were performed using tools provided in the SoftWorx v 3.5.1 software package (Applied Precision, Inc.). Mice and immunohistochemistry Min and wild-type littermate controls were obtained from Jackson Immuno- Research Laboratories. Mice were housed and maintained according to approved University of California Davis animal protocols. After killing, intestines were removed, ﬁxed as Swiss rolls in 4% formalin overnight, and embedded in parafﬁn. Parafﬁn-embedded tissue were cleared and re- hydrated according to standard protocols. Tissues were placed in CitraPlus antigen retrieval (Biogenix), heated in a microwave for 4 min, allowed to cool for 1 min, heated for 4 min, cooled for 1 min, and heated for 4 min. Room-temperature slides were rinsed twice in TBS-Tween. Tissues were blocked for 1 h in 50 mM TBS, pH 7.5, 0.1% Tween, 1% BSA, and 2.5% horse and goat serum (Vector Laboratories). Primary antibody was diluted in blocking buffer and incubated for 1 h in a humid chamber and room temperature. Tissue was rinsed twice in blocking buffer. Secondary anti- bodies were diluted 1:100 in blocking buffer and incubated for 1 h. Tissues were rinsed twice in TBST and once in PBS. Tissues were stained with DAPI for 10 min, rinsed in PBS, and mounted. FISH Bacterial artiﬁcial chromosomes containing deﬁned portions of the mouse genome were obtained from the BACPAC Resources Center (http://bacpac .chori.org; APC, RP24-73J15; Trp53, RP23-150N14; Tex11, RP24-140K17). FISH was preformed as described previously (Thatcher et al., 2005). The probe mixture consisted of nick-translated labeled probe (digoxigenin- labeled dUTP; Roche) and biotin-labled dUTP (Roche) in LSI/WCP hybridi- zation buffer (Vysis, Inc.) and was denatured on the tissue for 2.5 min at 85°C. When a single biotin-labeled probe was used, the membrane dye FM4-64 (Invitrogen) was incubated at 1:1,000 for 10 min just before add- ing the DAPI stain and coverslipping the slides. We noticed variability between discrete regions/crypts, suggesting that the condition of the tissue inﬂuences hybridization. To determine hybridi- zation efﬁciencies of probes using FISH, the following criteria were used: probe signals were required to be within 1.2 μm of in focus DAPI signal, and the total separation allowed between all probe signals was 6 μm. Nuclear diameter was measured at z-section with the largest diameter. Image and statistical analysis Analysis of spindle anchoring, anaphase, and furrow initiation parameters were performed on >100 videos for each cell line presented (i.e., control and APC1–1,450). Videos in which one parameter could not be determined were discarded (32 videos discarded). Linear regression was used to deter- mine the statistical difference between wild-type and Min spindle orientation. A t test was used for all other analysis. Online supplemental material Fig. S1 is an analysis of binucleate and multinucleate cells in a series of APC mutants and in several colon cancer cell lines. Fig. S2 is an analysis of anaphase timing in wild-type and APC mutant cells, and Fig. S3 presents an expanded analysis of anaphase and spindle histories of cells express- ing APC1–850. Fig. S4 provides more detailed views of mitotic cells in intes- tinal crypts and of the associated mitotic defects found in Min mice. In Fig. S5, to validate the carboxy-terminal APC antibody, we provide immunoblot analysis. We also provide evidence that the expression of stabilized β-catenin does not contribute to the accumulation of binucleate and multi- nucleate cells. Videos 1 and 2 show chromosome behavior in a mitotic control cell (Video 1) and in a mitotic APC1–1,450 cell (Video 2). Videos 3 and 4 show EB1-GFP dynamics in control mitotic cells (Video 3) and in APC1–1,450 mitotic cells (Video 4). Videos 5 and 6 show mitotic spindle be- havior in control cells (Video 5) and in APC1–1,450 cells (failed cytokinesis). Video 7 shows mitotic spindle behavior in APC1–1,450 cells (successful cyto- kinesis). Online supplemental material is available at http://www.jcb .org/cgi/content/full/jcb.200703186/DC1. We would like to thank the following for technical support: Alexander Borowsky, Larry Young, and Lisa Dillard-Temp for help with tissue preparation and histology; Janine LaSalle and Karen Thatcher for assistance with FISH; and Laurel Beckett and Qilu Yu for statistical analysis of mitotic orientation. We thank members of the Kaplan laboratory, the University of California Davis Cytoskeletal Club, and Dr. Bement for important feedback and input on the manuscript. This work was initiated with funding from a Sidney Kimmel Cancer Foundation grant to K.B. Kaplan and was continued with support from American Cancer Society grants (RSG-02-035-01 and RSG-02-035-05- CCG) and a California Coordinating Committee on Cancer grant to K.B. Kaplan. R.A. Green was funded by a Molecular and Cellular Biology training grant. APC MUTANTS LEAD TO TETRAPLOID CELLS IN VIVO • CALDWELL ET AL. 1119