Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited -- Potapova et al. 22 (8): 1191 Data Supplement - Supplemental Materials -- Molecular Biology of the Cell

Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited
Mol. Biol. Cell Potapova et al. 22: 1191

Supplemental Materials

This article contains the following supporting material:

Supplemental Materials
Movie01 - Cdk1 inhibition in prophase causes return to interphase that is fully reversible. This video shows Xenopus S3 cell that expresses alpha tubulin–GFP. This cell was treated with the Cdk inhibitor, Flavopiridol, during prophase. The Wee1/Myt1 inhibitor, PD 0166285, was added to prevent Cdk1 inhibition by phosphorylation. After Flavopiridol addition, the cell stopped progression into mitosis, the chromosomes de-condensed and the cell returned to an interphase morphology. Flavopiridol was washed out at 1 hour, and the cell re-entered mitosis and progressed through mitosis normally. Time is indicated as hours:minutes after Flavopiridol addition. This video is the source for the images shown in Figure 1C.
Movie02 - Cdk1 inhibition in prometaphase causes irreversible mitotic exit. This video shows a Xenopus S3 cell expressing alpha tubulin-GFP that was treated with Flavopiridol in early prometaphase. The Wee1/Myt1 inhibitor PD 0166285 was added to prevent Cdk1 inhibition by phosphorylation. After treatment at time 0 the cell underwent cytokinesis without chromosome segregation, the chromosomes decondensed, the nuclear envelope reformed and an interphase array of microtubules appeared. Flavopiridol was washed out at 1 hour. However, the cell did not reenter mitosis indicating that it had advanced to a G1-like state. Time is indicated as hours:minutes after Flavopiridol addition. This video is the source for the images shown in Figure 1D.
Movie03 - Normal mitosis in an untreated HeLa cell stably expressing histone H2B-mCherry and transiently transfected with human cyclin B1-GFP. The video shows a sequence of events from prophase to telophase. Time is indicated as hours:minutes after initiation of imaging. This video is the source for the images shown in Figure 2A.
Movie04 - A HeLa cell expressing histone H2B-mCherry and human cyclin B1-GFP was treated with Flavopiridol at prophase. The cell reverted to an interphase morphology. Cyclin B-GFP persisted in the cell but was removed from the nucleus. Time is indicated as hours:minutes after Flavopiridol addition. This video is the source for the images in Figure 2B.
Movie05 - A HeLa cell expressing histone H2B-mCherry and human cyclin B1-GFP was treated with Flavopiridol at prometaphase. The cell underwent cytokinesis without chromatid segregation. Cyclin B-GFP concentrated on the spindle/chromosomes and dispersed in the cytoplasm disappeared gradually due to degradation as the cell exited mitosis. Time is indicated as hours:minutes after Flavopiridol addition. This video is the source for the images in Figure 2C.
Movie06 - A HeLa cell expressing histone H2B-mCherry and human cyclin B1-GFP was treated with Flavopiridol at metaphase. The cell segregates chromatids and undergoes cytokinesis. Cyclin B-GFP concentrated on the spindle/chromosomes and dispersed in the cytoplasm disappeared rapidly, much of it prior to anaphase, due to degradation as the cell exited mitosis. Time is indicated as hours:minutes after Flavopiridol addition. This video is the source for the images in Figure 2D.
Movie07 - Inhibition of Wee1/Myt1 at the end of S phase triggers rapid entry into mitosis. This video shows HeLa cells stably expressing fluorescent histone H2B-GFP that were synchronized by double thymidine block at the S/G2 border and then treated with the Wee1/Myt1 inhibitor, PD0166285. Addition of the inhibitor triggered mitotic entry. Many cells then divided normally. Time is indicated as hours:minutes after the addition of Wee1/Myt1 inhibitor. This video is the source for the images shown in Figure 4A.
Movie08 - Inhibition of Wee1/Myt1 and Cdc25 at the end of S phase causes mitotic collapase. This video shows HeLa cells stably expressing fluorescent histone H2B-GFP that were synchronized by double thymidine block at the S/G2 border and treated with the combination of the Wee1/Myt1 inhibitor PD0166285 and Cdc25 inhibitor NSC663284. Addition of the two drugs triggered mitotic entry followed by mitotic collapase. Time is indicated as hours:minutes after the addition of Wee1/Myt1 and Cdc25 inhibitors. This video is the source for the images shown in Figure 4B.
Movie09 - Inhibition of Wee1/Myt1 together with Cdc25 in asynchronously growing HeLa cells causes mitotic collapase. A HeLa cells stably expressing histone H2B fused to mCherry and alpha-tubulin fused to GFP was treated with a combination of Wee1/Myt1 inhibitor, PD0166285, and Cdc25 inhibitor, NSC663284, and followed by live cell imaging. Drugs were added to the medium 30 minutes before the initiation of imaging. Time is indicated as hours:minutes after the initiation of imaging. This video is the source for the images shown in Supplemental Figure 4A.
Movie10 - Inhibition of Wee1/Myt1 and Cdc25 in asynchronously growing RPE1 cells causes mitotic collapse. RPE-1 cell stably expressing histone H2B fused to GFP was treated with a combination of Wee1/Myt1 inhibitor, PD0166285, and Cdc25 inhibitor NSC663284, and followed by live cell imaging (phase contrast and GFP). Drugs were added to the medium 20 minutes before the initiation of imaging. Time is indicated as hours:minutes after the initiation of imaging. This video is the source for the images shown in Supplemental Figure 4B.
Movie11 - MastL knockdown causes defects in chromosome alignment, segregation and cytokinesis in PD0166285-treated cells. This video shows H2B-GFP HeLa cells that were treated with MastL siRNA, synchronized by double thymidine block at the S/G2 border and then treated with the Wee1/Myt1 inhibitor, PD0166285. During mitotic entry stimulated by Wee1 inhibitor defects in metaphase alignment and chromatid segregation were evident. One cell in the field died. Time is indicated as hours:minutes after the addition of Wee1/Myt1 inhibitor. This video is the source for the MastL siRNA images shown in Supplemental figure 6.