Demonstration of chromosomal instability screen using a kinase cDNA library. (A) Mitotic escape in NL-20 cells results in viable tetraploids. NL-20 cells were treated overnight with colcemid or DMSO. Cells were fixed and stained for flow cytometry. (B) Kinase overexpression screen identifies kinases that regulate chromosomal stability. NL-20 cells were transduced with kinase cDNA constructs and grown with or without puromycin selection for 4 d. Cells were fixed and stained for flow cytometry. The percentage of cells with increased ploidy was calculated. The table represents those kinases that showed a reproducible increased ploidy phenotype in both NL-20 and HBE135 cells. (C) FISH analysis verifies increased ploidy. NL-20 cells were infected with the indicated kinase cDNAs and fixed for FISH analysis after 4 d. Fixed nuclei were stained with a chromosome 8-specific probe and counterstained with propidium iodide. The table shows the percentage of cells with greater than two copies of chromosome 8 for the indicated kinases.

shRNA screen identifies kinases that regulate chromosomal stability. (A) Flow chart describing the screening of a kinase shRNA library for kinases whose knockdown causes an increase in ploidy. NL-20 cells were infected in quadruplicate in 96-well plates, and ploidy was assessed by flow cytometry 4 d postinfection. Primary hits were rescreened against three different cell lines to find the strongest hits shown in the table. (Right) Column indicates the number of unique shRNAs that showed a ploidy phenotype. (B) Z score distribution (>4N) for two of the replicate screens. Z scores relative to the plate median of the percentage of cells with greater than 4N DNA content were calculated for each data point in each of four replicate screens. The large blue markers represent shRNAs that scored in follow-up screens in additional cell lines. (Right) Z scores for these shRNAs are shown. (C) Correlation of increased ploidy with gene knockdown. HBE135 cells were transduced with the indicated shRNAs. After 4 d, mRNA was prepared and cells were fixed for flow cytometric measurement of DNA content. The level of mRNA was calculated by measuring the ratio of the indicated gene to GAPDH for the indicated shRNAs vs. the same ratio in control samples. For flow cytometry, the percentage of cells with greater than 4N DNA content is shown. scr, scrambled; sh, short hairpin.

Measurement of increased ploidy by FISH. (A) Example of shRNAs that increase ploidy as measured by FISH. NL-20 cells were infected with the indicated kinase shRNAs and fixed for FISH analysis after 4 d. A centromeric probe specific for chromosome 12 (red) was hybridized to nuclei, which were then counterstained with DAPI (blue). sh, short hairpin. (B) Quantitation of FISH analysis for chromosome 12. FISH analysis was carried out with the indicated shRNAs in NL-20 and HBE135 cells. The number of nuclei with greater than two copies of chromosome 12 for each shRNA was measured using CellProfiler software. For each shRNA, at least 50 nuclei were counted.

Knockdown of several kinases results in multipolar mitoses and aneuploidy. (A) Knockdown of kinases results in multipolar mitoses. Kinase shRNAs were transduced into HBE135 cells, and the cells were selected for 21 d. Cells were fixed for immunofluorescence and stained with antibodies against tubulin (green) and pericentrin (red). DNA was stained with DAPI (blue). (Left) Examples of multipolar mitosis phenotypes are shown. (Right) Graph shows the mean number of poles for each sample (n = 25 for each). *P < 0.05 by Student's t test. (B) Sustained knockdown of NME1 results in a progression from tetraploidy to aneuploidy. HBE135 cells were transduced with the NME1 shRNA and selected for 4 or 17 d. Cells were fixed for FISH analysis and stained with centromeric probes for chromosomes 8 and 12. The number of copies of these two chromosomes in each nucleus is shown. (Right) Number of copies of chromosome 8 in nuclei with four copies of chromosome 12.

Knockdown of kinases results in growth in soft agar. (A) Knockdown of IRAK4 and NME1 promotes growth in soft agar. HBE135 cells were transduced with scrambled shRNAs or two shRNAs targeting IRAK4 or NME1. sh, short hairpin. Cells were selected for 21 d, seeded in soft agar, and incubated for an additional 21 d. Colonies were stained with crystal violet. (B) Soft agar growth requires sustained knockdown of NME1. HBE135 cells were transduced with NME1 shRNA and plated in a soft agar matrix at 4 or 17 d postinfection. The colony number is the average of three (4 d) or two (17 d) wells ± SEM. (C) Soft agar colonies are aneuploid. A single colony was picked from soft agar in cells containing the NME1 shRNA. The colony was expanded for several passages, and the cells were fixed for FISH analysis as described in Fig. 4B.