Reversibility of mitotic exit requires preservation of Cyclin B. a, A Xenopus S3 cell treated with 100 ng/ml nocodazole at time 0. At 30 min after nuclear envelope breakdown, nocodazole was removed and the cell was arrested at metaphase with MG132. Flavopiridol at 5 μM was added at 1:04:00 and removed 25 min later after mitotic exit. The complete video sequence is available as Supplementary Video 6. Bar = 10 μm. b, Experimental protocol employed for producing samples from Hela cells at various stages of induction and reversal of mitotic exit. c, Samples were analyzed for mitotic index and blotted for the indicated proteins.

Primary cultures of human cells can undergo mitotic exit reversal. An MG132-arrested metaphase cell was imaged by phase contrast microscopy after addition of 7.5 μM Flavopiridol at time 0. At 26 min, the Flavopiridol was removed. The complete video sequence is available as Supplementary Video 10. Bar = 10 μm.

The Cdk inhibitor Flavopiridol induces reversible mitotic exit and cytokinesis if proteasome activity is inhibited. a, Treatment of mitotic cells with Flavopiridol induces premature mitotic exit and cytokinesis without chromatid separation. A Xenopus S3 cell expressing GFP-α-tubulin was treated with 5 μM Flavopiridol at time 0. The black arrow indicates a chromosome not yet at metaphase when Flavopiridol was added. The white arrowhead indicates the reassembled nuclear envelope. The complete video sequence is available as Supplementary Video 1. Time is indicated as hours:minutes:seconds (a.- c.). b, Flavopiridol induces mitotic exit in cells arrested at metaphase with MG132. An MG132-arrested cell was treated with 10 μM Flavopiridol at time 0. The complete video sequence is found as Supplementary Video 2. c, Flavopiridol-induced mitotic exit and cytokinesis are reversible if the proteasome is inhibited. An MG132-arrested cell was treated with 5 μM Flavopiridol at time 0. The Flavopiridol was removed at 25 min. The complete video sequence is available as Supplementary Video 3. d, Flavopiridol induces normal mitotic exit changes in the distributions of Aurora B kinase and MKLP1 that are reversible. Xenopus S3 cells were incubated in medium containing MG132 and were fixed before and after treatment with 5 μM Flavopiridol. Samples were labeled for Aurora B kinase (upper panels) or MKLP1 (lower panels). Initial and final images (0 min and 60 min) were scaled identically for brightness and contrast. Others were scaled individually. Bars = 10 μm.

Cells expressing non-degradable Cyclin B1 undergo mitotic exit reversal. a, Hela cells expressing non-degradable cyclin B1 can undergo mitotic exit reversal without segregating chromosomes. Flavopiridol was added at time 0 and removed at 25 min. The complete video sequence is available Supplementary Video 7. b, Hela cells expressing non-degradable cyclin B1 can undergo mitotic exit reversal after chromatid separation. Flavopiridol added and removed as in (a). The complete video sequence is available as Supplementary Video 8. c, Hela cells expressing wild type Cyclin B1 do not undergo reversal of mitotic exit. Flavopiridol added and removed as in (a). The complete video sequence is available as Supplementary Video 9. Insets show GFP fluorescence images at time 0. Level of the wild type Cyclin B1 in (c) was approximately twice that of the non-degradable Cyclin B1 expressed in (a) and (b). d, Cells expressing high levels of non-degradable Cyclin B1 were more likely to undergo mitotic exit reversal. Cells that did not separate chromatids upon treatment with Flavopiridol are depicted by black symbols. Those that separated chromatids are depicted in red. Two cells that recondensed their chromosomes but had not opened their cleavage furrows by 90 min after Flavopiridol removal are depicted by hollow red symbols. Error bars show S.E.M. e, A mitotic Hela cell at metaphase (arrow) expressing non-degradable Cyclin B1, shows low levels of Cyclin A expression by immunofluorescence. Bars = 10μm.