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. 2016 Jul 2;5:e16270. doi: 10.7554/eLife.16270

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© 2016, Fong et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 5. — (A) 53BP1 normally disassociates from the kinetochores during anaphase, but can do so prematurely in prometaphase upon mitotic stress/delay. Immunofluorescence images of centrosomal (left) or acentrosomal (right) cells going through mitosis stained with the indicated antibodies. Bright BubR1 signals in prophase and prometaphase indicate SAC activation. Scale bar, 5 μm. (B) Disassociation of 53BP1 from mitotic kinetochores in cells experiencing mitotic stress/delay is time dependent. Quantification of proportion of mitotic cells with 53BP1 localization at the kinetochores. Cells arrested in G2/M were released into mitosis without centrosome (left), or with the spindle poison Eg5 inhibitor (right) as indicated, and then harvested at various time points after the release. Cells were stained with DAPI and antibodies against 53BP1 and BubR1 for scoring. Data are means ± SD. n>130, N = 3. (C) Disassociation of 53BP1 from kinetochores during mitotic stress is not reversible. Cells arrested in metaphase with MG132 for 4 hr followed by reactivation of SAC with nocodazole treatment for 10 min were stained with the indicated antibodies. Bright BubR1 or Mad2 signals indicate SAC activation. Scale bar, 5 μm. (D) 53BP1, USP28 and p53 form large nuclear foci in response to mitotic delay. PLK4^as; p21^-/- cells proliferating with or without 3MBPP1 stained with the indicated antibodies. Scale bar, 5 μm. (E) SAC is inactive in MPS1^as cells treated with 3MBPP1. MPS1^as cells treated with 3MBPP1 and Cdk1 inhibitor (RO-3306) for 18 hr were released into mitosis and were processed for immunofluorescence to visualize BubR1 and CREST. Note that SAC activation was disabled by MPS1 inhibition (weak BubR1 signals). Scale bar, 5 μm. (F) MG132 treated cells arrest in G1 in the absence of SAC activity. BrdU incorporation assay showing proportion of proliferating MPS1^as or MPS1^as; 53BP1^-/- cells, with or without 3MBPP1, following release into mitosis with different length of MG132 treatment. Data are means ± SD. Percentages are normalized to the untreated control. n>250, N = 3. (G) Model of 53BP1 and USP28 transducing mitotic stresses into p53 stabilization and p21 dependent cell cycle arrest. (H) A model proposing that the independent collaboration of SAC and 53BP1/USP28 drives efficient mitosis and cell fitness.

DOI: http://dx.doi.org/10.7554/eLife.16270.016

Figure 5—figure supplement 1. — (A) 53BP1 normally disassociates from the kinetochores during anaphase, but can do so prematurely in prometaphase upon mitotic stress/delay. Immunofluorescence images of centrosomal (left) or acentrosomal (right) cells going through mitosis stained with the indicated antibodies. Bright BubR1 signals in prophase and prometaphase indicate SAC activation. Scale bar, 5 μm. (B) Disassociation of 53BP1 from mitotic kinetochores in cells experiencing mitotic stress/delay is time dependent. Quantification of proportion of mitotic cells with 53BP1 localization at the kinetochores. Cells arrested in G2/M were released into mitosis without centrosome (left), or with the spindle poison Eg5 inhibitor (right) as indicated, and then harvested at various time points after the release. Cells were stained with DAPI and antibodies against 53BP1 and BubR1 for scoring. Data are means ± SD. n>130, N = 3. (C) Disassociation of 53BP1 from kinetochores during mitotic stress is not reversible. Cells arrested in metaphase with MG132 for 4 hr followed by reactivation of SAC with nocodazole treatment for 10 min were stained with the indicated antibodies. Bright BubR1 or Mad2 signals indicate SAC activation. Scale bar, 5 μm. (D) 53BP1, USP28 and p53 form large nuclear foci in response to mitotic delay. PLK4^as; p21^-/- cells proliferating with or without 3MBPP1 stained with the indicated antibodies. Scale bar, 5 μm. (E) SAC is inactive in MPS1^as cells treated with 3MBPP1. MPS1^as cells treated with 3MBPP1 and Cdk1 inhibitor (RO-3306) for 18 hr were released into mitosis and were processed for immunofluorescence to visualize BubR1 and CREST. Note that SAC activation was disabled by MPS1 inhibition (weak BubR1 signals). Scale bar, 5 μm. (F) MG132 treated cells arrest in G1 in the absence of SAC activity. BrdU incorporation assay showing proportion of proliferating MPS1^as or MPS1^as; 53BP1^-/- cells, with or without 3MBPP1, following release into mitosis with different length of MG132 treatment. Data are means ± SD. Percentages are normalized to the untreated control. n>250, N = 3. (G) Model of 53BP1 and USP28 transducing mitotic stresses into p53 stabilization and p21 dependent cell cycle arrest. (H) A model proposing that the independent collaboration of SAC and 53BP1/USP28 drives efficient mitosis and cell fitness.

DOI: http://dx.doi.org/10.7554/eLife.16270.016