Fig. 1. Induced ectopic expression of MAP4 results in a mitotic block characterized by monoastral spindles. (A) Immunoblots of cellular lysate, separated by 12% SDS-PAGE, using the indicated antibodies for detection. K562 cell lines harboring pMEP-vector-Co or pMEP-MAP4 were analyzed after various times of Cd²⁺-induced expression from the hMTIIa promotor. Arbitrary quantification was obtained from serial dilutions of cell lysates, which revealed tenfold increased expression of MAP4 after 24 hours and non-significant alterations in endogenous tubulin levels (see Relative tubulin amount). In the right-hand panel, cell lysates were separated by 8% SDS-PAGE to resolve slowly migrating MAP4 phosphoisomers characteristic of mitotic cells. (B) Transfected cells were Cd²⁺-induced for 20 hours, fixed and stained with anti--tubulin (green) and propidium iodine. A confocal section of a normal spindle (Vector-Co) and representative monoastral spindles caused by either MAP4 overexpression (MAP4) or by the Eg5 inhibitor monastrol (68 µM, 20 hours) are shown. The distribution of DNA content within transfected or monastrol-treated cell populations is also shown. (C) Cells induced to overexpress MAP4 as in panel B were fixed in methanol and stained with anti--tubulin (green) and anti-pericentrin (red). A confocal section of a representative monoastral spindle observed among MAP4-overexpressing cells is shown (bar, 6 µm). (D) K562, Jurkat and DG75 cell lines harboring pMEP-vector-Co or pMEP-MAP4 were induced for 20 hours with Cd²⁺ and mitotic figures were evaluated by epifluorescence microscopy with respect to numbers of spindle poles in cells double stained for DNA and MTs (n=450 cells). Data represent mean of two independent determinations.