Fig. 5. Depletion of CHO1/MKLP-1 causes multinucleation. (A) Efficient depletion of CHO1/MKLP-1 with vector-based RNAi. HeLa cells were transfected with either control vector or pBS/U6-CHO1. Two days after transfection, cells were harvested and cell lysates were subjected to direct western blot using antibodies indicated on the left. (B) HeLa cells were cotransfected with pBS/U6-CHO1-1st half, GFP and pBabe-Puro (control), with pBS/U6-CHO1, GFP and pBabe-Puro (CHO1 depletion) or with pBS/U6-CHO1, GFP-hamster-CHO1 and pBabePuro (CHO1 depletion, then rescue) at the ratio of 5:5:1. After 2 days of puromycin selection of transfection-positive cells, cells were further incubated up to day 6 in the presence of puromycin. Cell numbers were counted with a cytometer. (C) FACS profiles of CHO1-depleted cells at 3 days and 4 days after transfection. (D) Representative confocal images of CHO1-depleted cells. Blue indicates DNA staining with DAPI and green indicates -tubulin staining. (E) Quantification of results of three independent experiments (400 cells each); bars indicate standard deviations. (F-H) CHO1-depleted cells have multiple centrosomes. (F) HeLa cells were transfected as described in (B). After 2 days of puromycin selection, cells were stained with antibodies against -tubulin (green) and -tubulin (red), and DNA was stained with DAPI (blue). (G) Quantification of results indicates that almost every multinucleate cell has multiple centrosomes. (H) CHO1/MKLP-1-depleted cells form multiple spindle poles during mitosis. Scale bar, 10 µm.