Requirement of a Centrosomal Activity for Cell Cycle Progression Through G1 into S Phase Edward H. Hinchcliffe, Frederick J. Miller, Matthew Cham, Alexey Khodjakov, and Greenfield Sluder

Supplementary Material

Supplemental Figure 1. (a) Living BSC-1 cell as seen by phase contrast optics; the centrosome is at the center of the mass of granules next to the nucleus. (b) Another BSC-1 cell fixed and immunostained with antibodies against a-tubulin and g-tubulin. a-tubulin (green) and g-tubulin (red) signals are superimposed upon the phase contrast image. The slight separation of the centrosome from the nucleus makes this a favorable material for microsurgery. Bar = 5 mm.

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Supplemental Figure 2. Distribution of Golgi in a karyoplast and control cells. (a-f) Karyoplast (black arrow) going through mitosis and exiting mitosis as a single cell. (g-h) Same field with this karyoplast seen in phase contrast and fluorescence for bodipy FL ceramide. The fluorescence intensity in the perinuclear region of the karyoplast is similar to that seen in the control cells. Also, note that the distribution of Golgi in the control cells is not restricted to just the centrosomal region, and thus, the microsurgery used to produce karyoplasts would not remove all of it. Hours and minutes after the surgery are shown in the lower corner frames (a-f).

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Supplemental Figure 3. Mitosis and interphase arrest for a karyoplast (arrows) that does not cleave. The other cell is an uncut control. (a-b) The karyoplast flattens and resumes motility after surgery. (c-d) The control cell enters mitosis. (e-g) Mitosis in the karyoplast. The cleavage furrow retracts and the karyoplast exits mitosis as a single cell. (h-l) Post-mitotic karyoplast arrests in interphase until observations are terminated at 63 hours. Hours and minutes after microsurgery are shown in the lower left corner of each frame. Phase contrast optics. Bar = 10 mm.

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Supplemental Figure 4. Duration of mitosis for uncut control cells, karyoplasts, and control cells treated with 200-500 nM Taxol. Minutes between nuclear envelope breakdown and nuclear envelope reformation are shown along the horizontal axis. Each point represents the duration of mitosis for an individual cell. Arrowheads mark the means for each class. All control and Taxol-treated cells represented here divided at least two times.

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Supplemental Figure 5. Post-mitotic karyoplast does not contain centrioles. (a-d) Karyoplast completes mitosis and enters interphase as a single cell. (e) This karyoplast just before fixation 30 hours after the microsurgery. (f) Representative section (of 59 serial sections) of the same karyoplast; sections through the MTOC region did not contain any centrioles. Bar = 9.7 mm.

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