LEGENDS TO SUPPLEMENTAL FIGURESNature 2005-01-01023

Supplementary Figure 1. Characterization of viable cultures of diploid and tetraploid p53-/-MMECs. a, Control DMSO-treated sample for the DCB treated sample shown in Fig. 1a.Top,FACS profile of DMSO treated MMECs after 20 hrs. Bottom, fluorescence image showing cytokeratins (green) and DAPI-labeling of DNA (blue). Scale bars, 10 m. b, Multiple centrosomes and multipolar mitoses intetraploid MMECs. Labeling with a centrosome marker (pericentrin antibody, red) and DAPI (blue). Cells were treated with DCB for 18hr, washed into fresh medium for 2 hr, fixed, and labeled. 59% of cells exhibited multiple centrosomes (n=309/520). 53% of mitotic cells with multiple centrosomes were bipolar (top); 47% of these cells were mutipolar (bottom). Scale bars, 10 m. c, Robust growth of diploid and tetraploid p53-/-cultures. Phase contrast images of diploid (top) and tetraploid (bottom) MMECs at the indicated times after FACS-sorting. Scale bars, 20 m. Note: Immediately after FACS sorting we noted a small sub-G1 peak in both the diploid and tetraploid cultures that was not evident in later cultures. d, RobustBrdU incorporation (24 hr labeling, green) of both diploid (left) and tetraploid (middle) p53-/- MMECs 24 hrs after isolation by FACS. Centrosomes (pericentrin) are shown in red. Scale bars, 10 m.e, The diploid culture spontaneously develops a significant population of tetraploid cells after passage in culture. The distribution of chromosome numbers per cell in the diploid-derived culture after 10 passages (karyotypes from 100 cells).

Supplementary Figure 2. Tetraploid p53+/+ MMECs do not proliferate in vitro. a, FACS analysis of p53+/+ MMECs treated with DMSO (top) and DCB 2µM (bottom). The size of the 8C peak after 20 hr of DCB treatment is comparable to that of the p53-/- cultures (Fig. 1a), indicating that cytokinesis is blocked to an equivalent degree in both cultures. b, FACS-sorted diploid p53+/+ MMECs are able to proliferate in culture whereas tetraploid MMECs cannot. FACS profiles of sorted diploid and tetraploid MMECs after 96 hr culture (left), and phase contrast images of the same cells at the indicated times of culture (middle and right). Scale bars, 20 m. c, The percentage of cells incorporating BrdU after a 24 hr culture and labeling was done as in Supplementary Fig. 1d.

Supplementary Figure 3. Genome-wide array-CGH analysis of tetraploid p53-/-MMECs hybridized to diploid MMECs. The cells were isolatedby FACS sorting and immediately processed for array-CGH. The small signal fluctuations are statistically insignificant and are due to noise in the procedure.

Supplementary Figure 4. DCB does not induce DNA damage in p53-/- MMECs or wild-type p53+/+MMECs. a, -H2AX labeling of vehicle-treated and DCB-treated p53-/- MMECs. Freshly isolated organoids were plated onto 4-well culture slides and then were treated with DCB for 18hr. 1hr after washing out the DCB, -H2AX staining (07-164, Upstate) and in a parallel culture BrdU labeling was performed as in Wang, X. et al, Mol. Cell Biol., 2004, 24:5850-62 and in Wong, C. and Stearns, T. Nature Cell Biol., 2003, 5:539-44, respectively. The percentage of cells containing multiple foci are indicated below each image. For this experiment, the DMSO-treated sample serves as a negative control and the doxorubicin-treated sample serves as a positive control. More than 800 cells were scored for each sample by two independent observers. Each panel shows a montage of cells with representative patterns of labeling. Each experiment was performed twice with similar results. For the DMSO control sample, 19% of cells were in S-phase by BrdU-labeling and 23% of the DCB-treated cells were in S-phase (not shown). b, -H2AX labeling of vehicle-treated and DCB-treated p53+/+ MMECs (18 hr.). For the control DMSO sample, 22% of cells were in S-phase by BrdU-labeling and 18% of the DCB-treated cells were in S-phase. c, Absence of p53 induction by DCB in p53+/+ MMECs. Left, immunofluorescence of p53+/+ MMECs treated with DCB for 18hr as compared with positive and negative controls. p53 was detected with mAb 1C12 from Cell Signaling Technology, Inc. Right, Western blot of the same samples.

Supplementary Figure 5. DNA damage assessed by -H2AX labeling in p53-/- MMECs before and after FACS sorting. DCB treated samples (18 hr) were trypsinized, and either directly attached to coverslips for 22 hrs (pre-FACS) and labeled with anti-H2AX Ab or BrdU or FACS sorted followed by attachment and labeling. Each panel shows a montage of cells with representative patterns of labeling. a, Pre-FACS sample labeled for-H2AX as in Supplementary Figure 4. b, Diploid p53-/- MMECs after sorting labeled for-H2AX. c, Tetraploid p53-/- MMECs after sorting labeled for-H2AX. No significant difference in -H2AX labeling was detected either between the diploid and the tetraploid cells or before and after FACS sorting. We do see a modest increase in the number of cells with -H2AX foci (approximately 4% increased to 10%) immediately after trypsinization and re-plating relative to the sample labeled before trypsinization (see Supplementary Figure 4). We attribute this effect to the stress experienced by primary mammary epithelial cells transiently loosing their normal cell-cell contact.

Supplementary Figure 6. Gross chromosomal rearrangements in tetraploid-derived transformed cells growing in soft agar (Figure 2). GCR is evidenced by the nonreciprocal translocations (some recurrent) seen in 19 of 21 cells analyzed. a, Spectral karyotyping from a representative cell (NRTs indicated by arrows). Scale bar, 10 m. b, Table summarizing the SKY data from 21 cells.

Supplementary Figure 7. Characterization of the spontaneous tumors derived from tetraploid p53-/- MMECs. Immunohistochemical analysis of tumors indicates that the tumors are derived from myoepithelial cells. Labeling (brown) with an anti-pan cytokeratin mAb (left), an anti-keratin 14 Ab that is specifically expressed in myoepithelial cells (middle), and an anti-smooth muscle actin Ab (SMA, right). Co-stain is hematoxylin. Anti-Keratin 14 and anti-SMA labeling was positive in greater than 60% of cells in each tumor. Scale bars, 100 m. Immunohistochemistry suggested that these tumors were derived from myoepithelial cells, one of the two epithelial cell types in the breast.

Supplementary Figure 8. Gross chromosomal rearrangements in the spontaneous tumors derived from tetraploid p53-/-MMECs. a, Chromosome numbers in cells from two tumors. Although chromosome number varied widely in the tumor cells, the average number was near the 80 chromosomes of tetraploid cells. b, Summary of SKY data from twenty cells from two different tumors.

Supplementary Figure 9. Genome-wide array-CGH analysis of 8 tumors derived from tetraploid p53-/- MMECs. Note all tumors contain a 0.9 Mb amplicon containing the tandem cluster of 10 MMP genes on chromosome 9. Additionally, tumors 1, 2 and 6 contain an approximately 1.6 Mb amplicon containing the c-Met gene (2-4 fold amplification) on chromosome 6.

Supplementary Figure 10. Genes on the amplicons identified from tetraploid-derived tumors. a, Amplicon containing the MMP cluster. b, Amplicon containing c-Met.

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