Conservation of Relative Chromosome Positioning in Normal and Cancer Cells  Luis A. Parada, Philip G. McQueen, Peter J. Munson, Tom Misteli  Current Biology  Volume 12, Issue 19, Pages 1692-1697 (October 2002) DOI: 10.1016/S0960-9822(02)01166-1

Figure 1 Pairing of Two Translocated Chromosomes in Mouse Lynphoma Cells (A) Metaphase spread of an AT-13 cell showing one normal chromosome 12 (yellow), two normal chromosomes 15 (red), one del(14) (green; D14), one T(12;14) (T12), and one T(14;15) (T14). DAPI counterstaining is in blue. (B) An AT-13 interphase cell nucleus analyzed by FISH with WCP 12 (red), 14 (blue), and 15 (green). In single focal planes by wide-field microscopy, chromosome translocations 12:14 (T12) and 14:15 (T14) are visible as double color red-blue and blue-green signals, respectively. The two translocated chromosomes are frequently positioned in close proximity (arrow). The scale bar represents 2 μm. (C) 3D reconstruction of an AT-13 interphase cell nucleus showing the proximal association between T12 and T14. (D) Histograms of nearest neighbors. T12 and T14 frequently form a proximal pair. (E) Statistical analysis of nearest-neighbor data. The observed frequencies of chromosome pairing were compared to expected values (solid lines) ± standard deviation (dashed line) by Chi-square analysis of contingency tables. Expected values were calculated under the assumption that chromosomes paired randomly. (F) Interchromosome distance measurements for chromosomes 12, 14, T12, and T14. The median distance is indicated with a solid line and is significantly lower for T12-T14 than for any other chromosome pair. Each symbol represents a single chromosome pair. (G) Statistical analysis of interchromosome distance measurements. The observed frequencies of chromosome pairing were compared to expected values (solid lines) ± standard deviation (dashed line) by Chi-square analysis of contingency tables. Random pairing of chromosomes was assumed for the calculation of expected values. Current Biology 2002 12, 1692-1697DOI: (10.1016/S0960-9822(02)01166-1)

Figure 2 A Chromosome 12, 14, 15 Cluster in Normal Cells Normal mouse splenocyte nuclei were analyzed by FISH with WCP 12 (red), 14 (blue), and 15 (green). In single focal planes by wide-field microscopy, this subset of chromosomes forms a triplet cluster (arrows) in interphase of (A, B) diploid and (C) two clusters in tetraploid cells. The scale bar represents 2 μm. (D, E) Chi-square analysis of contingency tables was used for comparing the frequencies of 12-14-15 cluster formation versus all other kinds of triplets of visualized chromosomes. (D) Nearest-neighbor analysis. (E) Interchromosome distance measurements. The 12-14-15 cluster is significantly more frequent than any other triplet combination of this subset of chromosomes. (F, G) Chi-square analysis of contingency tables was used for comparing frequencies of 12-14-15 cluster formation versus 1-12-14, 1-12-15, 1-14-15 clusters. (F) Nearest-neighbor analysis. (G) Interchromosome distance measurements. The 12-14-15 cluster is significantly more frequent than any cluster containing chromosome 1. Current Biology 2002 12, 1692-1697DOI: (10.1016/S0960-9822(02)01166-1)

Figure 3 Control for Cluster Formation (A) Normal mouse splenocytes and (B) AT-13 cells were analyzed by FISH with WCP 1 (red), 4 (blue), and 19 (green). This analysis detected no particular pattern of distribution of these chromosomes in single focal planes by wide-field microscopy. (C) Statistical analysis of pairing for chromosomes 1, 4, and 19 in normal splenocytes. Chi-square analysis of contingency tables was used for comparing the observed frequencies of chromosome pairing to expected values (solid lines) ± standard deviation (dashed line). Random pairing of chromosomes was assumed for the calculation of expected values. (D) Statistical analysis of cluster formation of 1, 4, and 19 in normal splenocytes. There was no significant difference between the chromosome triplet 1-4-19 formation and all other possible triplets of visualized chromosomes. (E) Statistical analysis of pairing of chromosomes 1, 4, and 19 in AT-13 cells. No preferential pairing among these chromosomes was detected. (F) Statistical analysis of triplet formation of 1, 4, and 19 in AT-13 cells. No evidence for cluster formation among these chromosomes was detected. The observed frequencies of chromosome pairing and cluster formation were compared to expected values (solid lines) ± standard deviation (dashed line). Current Biology 2002 12, 1692-1697DOI: (10.1016/S0960-9822(02)01166-1)

Figure 4 Maintenance of Relative Positioning of Chromosomes in Mitotic AT-13 Cells and Normal Splenocytes (A) Statistical analysis of pair formation of T12 and T14 in mitotic AT-13 cells. T12 and T14 were significantly more likely to form a proximal pair than any other two visualized chromosomes in metaphase AT-13 cells. The observed values were compared to expected values (solid lines) ± standard deviation (dashed line). (B) Statistical analysis of triplet cluster formation of chromosomes 12, 14, and 15 in normal mitotic splenocytes. Chromosomes 12, 14, and 15 were significantly more likely to form a cluster than any other combination of these three chromosomes. Current Biology 2002 12, 1692-1697DOI: (10.1016/S0960-9822(02)01166-1)