Electron microscopic analysis of U-2 OS/centrin-GFP cells either DMSO-treated with 0.1% DMSO (A) or treated with 1 μM Z-L₃VS for 48 h (B). Scale bars indicate 500 nm.

(A) Quantification of U-2 OS/centrin-GFP cells with more than one daughter centriole per maternal centriole at 48 h after transfection with empty vector (neo), cyclin E, CDK2 and PLK4 either alone or in combination. Each bar represents mean and standard error of at least three independent experiments with a minimum of 100 cells counted per experiment.
(B) Quantification of IMR-90 normal human fibroblasts with more than one daughter centriole per maternal centriole at 48 h after transfection with empty vector (neo), cyclin E, CDK2 and PLK4 either alone or in combination. Centrioles were visualized by immunofluorescence microscopy for centrin. Each bar represents mean and standard error of at least three independent experiments with a minimum of 50 cells counted per experiment.

(A) Fluorescence microscopic analysis of U-2 OS cells stably expressing centrin-GFP to visualize centrioles (arrows; inserts) after either treatment with 0.1% DMSO (top panels) or 1 μM Z-L₃VS (bottom panels) for 48 h. Nuclei stained with DAPI. Scale bar indicates 10 μm.
(B) Quantification of U-2 OS/centrin-GFP cells with more than one daughter centriole per maternal centriole after treatment with either 0.1% DMSO or 1 μM Z-L₃VS for 48 h. Each bar represents mean and standard error of at least three independent experiments with a minimum of 100 cells counted per experiment.

(A) Fluorescence microscopic analysis of mitotic U-2 OS/centrin-GFP cells. A normal bipolar metaphase is shown in comparison to a pseudobipolar metaphase cell and a multipolar (tripolar) anaphase cell. More than two images are merged for middle and right panels to show all centrioles. Chromosomes stained with DAPI.
(B) Quantification of mitotic abnormalities in U-2 OS/centrin-GFP populations after treatment with 0.1% DMSO or 1 μM Z-L₃VS for 24 h and incubation in normal media for an additional 24 h. Only dividing cells were evaluated. Each bar represents mean and standard error of three independent experiments with at least 100 mitotic cells counted per experiment.

(A) Immunoblot analyses of U-2 OS/centrin-GFP cells transfected with either control siRNA duplexes (control) or siRNAs targeting cyclin E1, cyclin E2, cyclin A2, CDK2 or PLK4 for the indicated time intervals. Immunoblots for actin are shown to demonstrate loading of equal amounts of protein.
(B) Fluorescence microscopic analysis of U-2 OS/centrin-GFP cells transfected with control siRNA duplexes (top panels) or siRNAs targeting cyclin E1 (bottom panels) following treatment with 1 μM Z-L₃VS for 48 h. Cells were co-transfected with DsRED fluorescent protein as transfection marker. Nuclei stained with DAPI. Scale bar indicates 10 μm.
(C) Quantification of U-2 OS/centrin-GFP cells transfected with the indicated siRNAs followed by either control treatment with 0.1% DMSO or 1 μM Z-L₃VS for 48 h. Each bar represents mean and standard error of at least three independent experiments with a minimum of 100 cells counted per experiment.

(A) Fluorescence microscopic analysis of U-2 OS/centrin-GFP cells transiently transfected with either empty vector (control) or HPV-16 E7 after 24 h or 48 h. Note the formation of two daughters at a single maternal centriole after 24 h in a HPV-16 E7-transfected cell (middle panels, insert). After 48 h, this pattern was less frequent and the majority of cells with abnormal centriole numbers showed a more dispersed arrangement (bottom panels, insert, arrows). Cells were co-transfected with DsRED fluorescent protein as transfection marker. Nuclei stained with DAPI. Scale bar indicates 10 μm.
(B) Quantification of U-2 OS/centrin-GFP cells with more than four centrioles in a random arrangement (gray bars) in comparison to cells with more than four centrioles and a concurrent formation of more than one daughter per maternal centriole (black bars). Cells were transfected with empty vector (control) or HPV-16 E7 for 24 h or 48 h. Each bar represents mean and standard error of at least three independent experiments with a minimum of 100 cells counted per experiment.
(C) Quantification of U-2 OS/centrin-GFP cells with more than four centrioles and concurrent formation of more than one daughter centriole at a maternal centriole after treatment with 1 mM HU for the indicated time intervals. dH₂O-treated cells are shown as controls. Each bar represents mean and standard error of at least three independent experiments with a minimum of 100 cells counted per experiment.