Fig. 1. An experimental system to visualize chromosome translocations in living cells.

(A) NIH3T3duo cells containing an integrated LacO- IScel array and three TetO-IScel-TetO arrays, stably expressing GFP-LacR and mCherry-TetR, respectively. Scale bar, 10 μm. (B) Colocalization of LacO (green) and TetO (red) arrays in NIH3T3duo cells 24 hours after expression of IScel. Scale bar, 10 μm. (C) Percentages of the cells with paired LacO and TetO arrays in indicated cell populations. Values represent means ± SD from at least three independent experiments (7500 to 16,500 cells with LacO and TetO analyzed per sample; *P < 0.05, **P < 0.001, ***P < 0.0001, Student t test or χ² test). (D) Real-time PCR analysis for detection of Lac-Tet translocations in NIH3T3duo cells transfected with ISceI or ISceID44A for the indi cated times. PCR was performed using primers located in the Lac orTet operator sequences. Standard curve was generated by spiking-in at the indi cated ratios with NIH2/4 cells, which contain an integrated LacO-IsceI-TetO array (4). Values are normalized to 1:2000 sample and represent means ± SD from three independent experiments. *P < 0.05, ***P < 0.0001, two-tailed Student t test. (E) Untreated cells or cells transfected for 24 hours with the indicated plasmids were fixed and stained with DAPI and uHTI was performed to assess the cell cycle status of individual cells (fi g. S3, A and B). The percentage of cells with paired LacO- TetO arrays was determined. Values represent means ± SD from three independent experiments; one-way analysis of variance (ANOVA) test or χ² test (ISceI positive: G₁, n = 3620; S, n = 985; G₂+M, n = 1295; ISceID44A positive: Gi, n = 1851; S, n = 1052; G₂+M, n = 891). (F) NIH3T3duo cells transfected for 24 hours with ISceI were stained with DAPI and sorted into G₁ or S+G₂+M populations (fig. S3E). Identical numbers of gated G₁, S+G₂+M, and asynchronous cells were used to extract DNA and perform real-time PCR. Values represent means ± SD from two independent experiments (P > 0.05, one-way ANOVA). (G) Real-time PCR for LacO-TetO translocations in NIH3T3duo cells transfected with ISceI for 24 hours and arrested in G₁ phase by contact inhibition or at the G₂/M boundary by treatment with nocodazole (fig. S3, A and F). Values represent means ± SD from three experiments (P > 0.05, one-way ANOVA).