FIG. 6.

Pin1-induced centrosome amplification results in aneuploidy and cell transformation. (A) Experimental scheme. NIH 3T3 cells were transfected with Pin1-myc or control vector and selected with G418 to generate pools of stable transfectants. After confirming the expression of Pin1-Myc in cells with anti-Myc antibodies, cells were arrested at the G₁/S boundary for 24 h with aphidicolin and released into fresh medium; this was followed by various assays. (B to D) Pin1 overexpression causes the formation of multipolar spindles. At each point following the release from cell cycle arrest, cells were fixed and costained with anti-α-tubulin and anti-γ-tubulin antibodies followed by DAPI staining. (B) Representative mitotic cells with bipolar or multipolar spindle poles were shown. (C) Cells containing more than two mitotic spindle poles were scored in 200 mitotic cells. A representative Pin1-myc transfected cell with three-directional cell division is shown in panel D. Error bars, standard deviations. (E and F) Pin1 overexpression induces aneuploidy. Stable NIH 3T3 cells were released from the aphidicolin block, cultured for the indicated number of passages and then harvested for metaphase spreads. Representative chromosome preparations are shown in panel E, and the chromosome number per cell was calculated from over 100 individual metaphase spreads in each group (F). (G and H) Pin1 overexpression induces cell transformation. Stable NIH 3T3 cells were released from the aphidicolin block and seeded on plastic plates for 3 weeks, followed by crystal violet staining (G), or cultured in soft agar for 3 weeks and the number of colonies formed per 1,000 cells was scored (H). Colony numbers are the means ± standard deviations of three independent experiments (right panel). Error bars, standard deviations.