Fig. 5. Nuf2- and Hec1-deficient cells show prometaphase arrest associated with aberrant chromosomes and spindles that lead to chromosome missegregation. (A) Chromosome morphology and -tubulin staining (green) of Nuf23-63 and Hec25-1 cells in the absence or presence of tet. DNA was counterstained with DAPI (blue). In the absence of tet, cells show the normal staining pattern for -tubulin (upper panels in both cell lines). In the presence of tet, chromosomes are not aligned at the metaphase plate. Arrows indicate misaligned chromosomes at the metaphase plate. Apoptotic cells were observed in both mutants 48 hours after addition of tet. We also detected cells with monopolar and multipolar spindles. (B) Quantitation of aberrant Nuf23-63 and Hec25-1 cells after inhibition of transgene expression following addition of tet at time 0. We scored the number of interphase cells, normal metaphase cells, aberrant metaphase cells, anaphase cells and apoptotic cells. Apoptotic cells were detected by TUNEL assay. We scored approximately 3000 cells for each time point. (C) To examine chromosome loss, we used FISH analysis with chromosome-specific painting probes. We used painting probes for chicken chromosomes 1 and 2. Because DT40 cells have three copies of chromosome 2, five painted chromosomes were observed in normal cells. Nuf23-63 and Hec25-1 cells were cultured after addition of tet. At the indicated times cells were treated with colcemid for 1.5 hours, and the number of painted chromosomes per cell was determined. The number of painted chromosomes was scored in approximately 1000 metaphase cells.