1. Volume 15, Issue 6, Pages 977-990 (September 2004)
Inhibition of DNA Decatenation, but Not DNA Damage, Arrests Cells at Metaphase 
Dimitrios A. Skoufias, Françoise B. Lacroix, Paul R. Andreassen, Leslie Wilson, Robert L. Margolis 
Molecular Cell 
Volume 15, Issue 6, Pages (September 2004)
DOI: /j.molcel

2. Figure 1
DNA Damage Imposed during Mitosis Does Not Impede Mitotic Exit
(A) Mitotic HeLa cells were collected by selective detachment after being blocked in mitosis with Noc (0.04 μg/ml) for 16 hr (Mitotic s/o). Cells were then released from Noc in medium (control release) or in the presence of VP-16 (5 μg/ml) or adriamycin (2 μg/ml), and samples for 2D FACScan analysis were taken 1 and 2 hr after the release. Alternatively, mitotic HeLa cells were exposed to 12 Gy γ irradiation and then collected by shake-off and released from Noc (12 Gy). Regardless of treatment, a portion of the cell population, comparable to controls, proceeded to G1 as determined by loss of MPM-2 signal (right dot-plot panel), and the majority have become 2N by 2 hr (left histogram panel). Percents shown indicate the number of total cells positive for MPM-2.
(B) DNA damage induces recruitment of γ-H2AX onto chromosomes. Asynchronous HeLa cells were exposed to VP-16, adriamycin, and 12 Gy γ irradiation for 2 hr and then fixed and stained for IF microscopy using anti-γ-H2AX antibodies and propidium iodide. Metaphases are shown.
(C) Cells with chromosomes still positive for γ-H2AX proceed through aberrant cytokinesis 2 hr after either 12 Gy γ irradiation or adriamycin treatment. Bar = 5 μm.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
Mitotic Exit in the Presence of DNA Damage Imposed in Mitosis Is Independent of p53 Status
(A) Mitotic U2OS, HCT116 p53+/+, and HCT116 p53−/− cells were collected by selective detachment (see Experimental Procedures) and exposed to adriamycin (2 μg/ml) following Noc release or 12 Gy γ irradiation before Noc release. Samples for 2D FACScan analysis were taken 1 and 2 hr after Noc release. The mitotic index was calculated relative to the mitotic value of cells at time 0, immediately following shake-off. Mitotic indices of cells exposed to DNA damaging agents were similar to untreated control cells 1 and 2 hr after Noc release.
(B) DNA damage in interphase leads to G2 delay. HCT116 p53+/+ cells were synchronized in G1/S phase with HU (2 mM, 16 hr) and then following release from HU were exposed to Noc (0.5 μg/ml) or to the combination of Noc (0.5 μg/ml) and either VP-16 (5 μg/ml), adriamycin (0.2 μg/ml), or 12 Gy γ irradiation. Samples for 2D FACScan analysis (as described in Figure 1) were taken 20 hr after treatment. Following DNA damage, cells blocked in G2 (MPM-2-negative 4N cells), whereas the majority of untreated cells blocked in mitosis.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
Mitotic Arrest Induced by ICRF-193 Is Independent of DNA Damage
(A) Mitotic HeLa cells were collected by selective detachment after being blocked in mitosis with Noc (0.04 μg/ml) for 16 hr. Cells were then released from Noc in the presence of ICRF-193 (100 μM) or in adriamycin (2 μg/ml), and samples for 2D FACScan analysis were taken 1 hr and 2 hr after the release. Inhibition of DNA decatenation by the topo II inhibitor ICRF-193 leads to stable mitotic arrest, in contrast to failure to arrest in mitosis following DNA damage imposed by adriamycin (see also Figure 1A).
(B) ICRF-193, unlike adriamycin, does not induce γ-H2AX on chromosomes. Unsynchronized HeLa cells were exposed to adriamycin (2 μg/ml) or to ICRF-193 (100 μM) for 2 hr and then fixed and stained for IF microscopy using anti-γ-H2AX antibodies and propidium iodide. The control is an untreated metaphase cell. Confocal microscope images were collected with the same microscope settings for all figure parts. Metaphases are shown. Bar = 5 μm.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
Lack of Decatenation Blocks Mitotically Synchronized HeLa Cells at Metaphase
HeLa cells were incubated overnight with 0.04 μg/ml Noc, and mitotic cells were then collected by shake-off and either released from Noc arrest into drug-free medium or medium containing ICRF-193. Alternatively, cells were exposed to 12 Gy γ irradiation before release into medium.
(A) Analysis of mitotic arrest. The percent of cells remaining in mitosis was quantitated by microscopic analysis of MPM-2 status or by 2D FACScan analysis at the time points indicated. γ-irradiated cells exited mitosis, yielding values comparable to controls. By contrast, ICRF-193 induced mitotic arrest, whether measured by cell counts or by FACScan quantitation. The starting number of cells in mitosis was 97% ± 2% (n = 300) following Noc shake-off.
(B) IF images of cells at 2 hr following release from Noc. ICRF-193-treated cells are mostly mitotic, and MPM-2 positive (green), and are in metaphase as determined by propidium iodide (PI) stain. γ-irradiated cells have exited mitosis 1 hr after release with aberrant cytokinesis, creating chromatin bridges.
(C) HeLa cells, 9 hr after release from double thymidine block (time = 0 hr), were treated or not with NOC, ICRF-193, and VP-16. FACScan analysis revealed that NOC-treated cells accumulated in mitosis. VP-16-treated cells passed through mitosis with the same timing as untreated cells, whereas ICRF-193 cells remained blocked in mitosis.
(D) ICRF concentration dependency of arrest. Cells synchronized in mitosis as in (A) were exposed to different concentrations of ICRF during release, as indicated. The percent mitotic was determined by flow cytometry of MPM-2 signal relative to the signal at time 0.
(E) VP-16 concentration dependency of arrest. Cells were exposed to different concentrations of VP-16, as indicated, during mitotic release and analyzed as in (D).
(F) Microscopic images of cells arrested at 2 hr after release in 10 μM ICRF-193 or 250 μg/ml VP-16, stained as in (B). All samples derived from double thymidine block (C) and Noc shake-off synchronization (A, D, and E) were run in parallel and were derived from the same starting population of cells.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5 Metaphase Arrest Due to ICRF-193 Is Bub1 Independent
(A) IF microscopy shows that Bub1 is not recruited to kinetochores in ICRF-193-arrested metaphase cells (left). By comparison, cells blocked in mitosis with Noc (center), or treated first for 1 hr with ICRF-193 and then with a combination of ICRF-193 and Noc (0.1 μg/ml), are strongly positive for Bub1 (right). Bub1 signal was collected using quantitative photon counting.
(B) Kinetochores (arrows) of metaphase chromosomes exhibit equivalent separation due to spindle tension (see insets, lower right, in microscopic images), as determined by quantitative measurement of interkinetochore distances. 77 and 73 visible kinetochores, from 15 untreated and 15 ICRF-193-treated cells, respectively, were measured.
(C) ICRF-193 induces a mitotic block in cells expressing ΔN-mBub1. H261-HeLa cells expressing the ΔN-mBub1in the absence of tetracycline were treated or not with ICRF hr after release from double thymidine block (indicated as time = 0 hr). ICRF-193-treated cells remained blocked for the next 4 hr, whereas untreated cells exited mitosis.
(D) Treatment with Noc shows H261-HeLa tet− cells have compromised Bub1 function. FACSscan analysis of H261-HeLa cells in the presence and absence of tetracycline were synchronized by double thymidine block and then treated with Noc. After 24 hr in Noc, ΔN-mBub1-expressing cells did not block in mitosis but entered a new round of mitosis as evidenced by the prominent 8N peak in contrast to tet+ cells which arrested in mitosis, as described (Taylor and McKeon, 1997).
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
ICRF-193 and VP-16 Do Not Induce Recruitment of Mad2 to Kinetochores but Arrest Is Mad2 Dependent
(A) IF microscopy of HeLa cells stably transfected with GFP-Mad2 shows Mad2 is not recruited to ICRF-193-arrested metaphase chromosomes (left), compared to positive signals in Noc-blocked mitotic cells (center) or to cells treated with a combination of ICRF-193 and Noc (right). All cells were imaged at the same confocal microscope settings. Bar = 5 μm.
(B) Quantitation of GFP-Mad2-positive kinetochores in ICRF-193-treated cells. Data were collected using quantitative photon counting. The percentage of cells with one or more GFP-Mad2-positive kinetochores was calculated for each of the treatments. At least 15 cells in each of the treatments were analyzed. The majority of asynchronous ICRF-193-treated cells, cells released from Noc into ICRF-193, or cells released from Noc for 2 hr were negative for GFP-Mad2 on kinetochores. In contrast, all Noc-blocked cells, and cells cotreated with both Noc and ICRF-193, contained GFP-Mad2-positive kinetochores. Bars indicate either no kinetochores are positive (gray) or one or more are positive (black) in a given cell.
(C) IF microscopy of GFP-Mad2-expressing HeLa cells, 9 hr after being released from double thymidine block and then treated with either Noc (0.04 μg/ml), ICRF-193 (100 μM), or VP16 (250 μg/ml) for 3 hr. All cells were imaged at the same confocal microscope settings.
(D) The percentage of cells with one or more GFP-Mad2-positive kinetochores was calculated for each of the treatments from cells prepared as in (C). Three sets of data of at least 25 cells/set of data were counted. The majority of metaphase cells of untreated and ICRF-193 (100 μM)- and VP16 (250 μg/ml)-treated cells were negative for Mad2. In contrast, all nocodazole-treated cells had kinetochores positive for GFP-Mad2. Almost all GFP-Mad2-positive cells in ICRF-193 or VP-16 had only one positive kinetochore, as shown for one ICRF-193-treated metaphase cell (C).
(E) Western blots of lysed cell extracts, showing cyclin B and securin levels at time 0 (shake-off) and after 2 hr of release (Rel) from Noc into medium alone (DME), or 2 hr of ICRF-193 exposure upon release, or 2 hr after release following 12 Gy γ irradiation. β-tubulin is a loading control.
(F) Mad2 is in a complex with Cdc20 in ICRF-193-blocked metaphase cells. Immunoprecipitates, using either Mad2 or Cdc20 antibody, were made using extracts of control synchronous mitotic cells, blocked with Noc and collected by shake-off, or of cells released from Noc arrest into ICRF-193 or into medium alone (DME) for 2 hr. Cdc20 immunoprecipitates, crossblotted with Mad2 antibody, show Mad2 is equivalently present in control mitotic and ICRF-193-blocked cells, but depleted in DME released cells that have exited mitosis. Western blots for Mad2 and Cdc20 from total extract are shown as loading controls.
(G) Extracts from untreated and Mad2 siRNA-transfected HeLa extracts, 48 hr after transfection (20 μg total protein loaded), were examined by immunoblot analysis to confirm suppression of MAD2 expression.
(H) Controls and cells transfected with nonspecific siRNA or Mad2 siRNA were exposed to ICRF-193 (100 μM) 9 hr after release from double thymidine block (time = 0 hr). MPM2 labeling from 2D FACScans shows that control and nonspecific siRNA-transfected cells accumulate in mitosis in the presence of ICRF-193, whereas Mad2 siRNA-transfected cells do not.
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7
Inhibition of Cdc2 by Roscovitine in ICRF-193-Treated Cells Leads to Mitotic Exit with Centromere Separation
HeLa cells, 9 hr after double thymidine block release, were incubated with ICRF-193 (100 μM) for 2 hr to accumulate cells in metaphase, and cells were induced to exit mitosis by coincubation with 100 μM roscovitine for 30 min. Fixed cells were then stained for IF microscopy with anti-tubulin, CREST autoimmune sera, or anti-lamin B antibodies (green) and counterstained with propidium iodide (PI; red). Roscovitine induced rapid mitotic exit in ICRF-193-treated cells as witnessed by the assembly of nuclear lamina surrounding chromatin. However, roscovitine treatment caused ICRF-193-treated cells to enter anaphase with normal centromere separation and with extensive chromosome bridges.
Molecular Cell  , DOI: ( /j.molcel )