1. Volume 44, Issue 4, Pages 597-608 (November 2011)
Aurora-B Mediated ATM Serine 1403 Phosphorylation Is Required for Mitotic ATM Activation and the Spindle Checkpoint 
Chunying Yang, Xi Tang, Xiaojing Guo, Yohei Niikura, Katsumi Kitagawa, Kemi Cui, Stephen T.C. Wong, Li Fu, Bo Xu 
Molecular Cell 
Volume 44, Issue 4, Pages (November 2011)
DOI: /j.molcel
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Figure 1 ATM Is Activated in an Aurora-B-Dependent Manner in Mitosis
(A and B) HeLa cells were synchronized by thymidine double block (A) or treated with nocodazole (B) for 16 hr before they were harvested. Total cell lysates were immunoblotted with indicated antibodies.
(C) Mitotic HeLa cells were collected by mitotic shakeoff. Endogenous ATM was immunoprecipitated and subjected to the in vitro kinase assay.
(D) HeLa cells transiently transfected with control or Aurora-B siRNA (with or without siRNA-resistant Aurora-B) were exposed to mock or nocodazole. Total cell lysates were immunoblotted with indicated antibodies.
Molecular weights shown are in kiloDalton (kD). See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Aurora-B Phosphorylates ATM on Ser1403 In Vitro
(A) Immunoprecipitated Aurora-B was incubated with recombinant proteins consisting of GST-fused peptides derived from the various regions of human ATM in the presence of 32P-labeled ATP.
(B) Immunoprecipitated Flag-tagged wild-type (WT) or kinase-dead (KD) forms of Aurora-B were incubated with GST-fused peptides containing aa 1350–1450 fragments of ATM (either WT or serine 1403 mutated to alanine).
(C) Phospho-(ATM-S1403p) or nonphospho-(ATM-S1403) peptides were incubated with the recombinant GST-His-Aurora-B protein in the presence of ATP.
(D) Sequence homology of ATM around Ser1403 in different species.
See also Figure S2.
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4. Figure 3 Aurora-B Mediates ATM Ser1403 Phosphorylation during Mitosis
(A) ATM Ser1403 is phosphorylated in response to nocodazole treatment. HeLa cells were treated with mock, IR (6Gy), or nocodazole and subjected to western blot with the indicated antibodies.
(B) EBV-transformed lymphoblast cell lines, possessing proficient (GM0536) or deficient (GM1526) ATM, were treated with nocodazole. Total cell lysates were treated with λ-phosphatase followed by western blot.
(C) HeLa cells were transfected with control or ATM shRNA followed by mock, nocodazole, or IR (6Gy) treatment.
(D–F) Exponentially growing HeLa cells were stained with β-tubulin, CREST, ATM S1403p, and DAPI.
(G) HeLa cells were stained with ATM S1403p (red), Aurora-B (green), and DAPI (blue). Two representative images are shown (Image 1 and Image 2).
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Aurora-B Phosphorylates ATM Ser1403 to Activate ATM in Mitosis
(A) Aurora-B is required for ATM Ser1403 phosphorylation in mitosis. HeLa cells, transfected with control or Aurora-B siRNA, were treated with mock or nocodazole and subjected to western blot with the indicated antibodies.
(B) HeLa cells were treated with the Aurora-B inhibitor, Hesperadin, in the absence or presence of nocodazole.
(C) ATM Ser1403 phosphorylation is required for mitotic ATM activation. Vector, WT, S1403A, or S1981A mutants of ATM were transiently transfected into 293T cells and treated with nocodazole. Exogenous proteins were then immunoprecipitated with an anti-Flag antibody followed by western blot using the indicated antibodies.
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6. Figure 5 ATM Deficiency Leads to an Impaired Spindle Checkpoint.
(A) Cells were treated with nocodazole and the mitotic index was measured by flow cytometric based Histone H3 S10p staining. The percentage of mitotic cells shown represents the averages of at least triplicate samples. Error bars represent the standard deviation.
(B) Cells were treated with nocodazole for 48 hr and stained with propidium iodide to measure DNA content. Percentages of cells with greater than 4N DNA content are shown. Error bars represent the standard deviations of at least triplicate samples.
(C) Generation of isogenic stable cell lines with control or ATM knockdown. HeLa cells were stably transfected with either control or ATM shRNA. ATM expression levels for control and ATM shRNA clones #1 and #2 are shown.
(D) The isogenic cell lines were treated with nocodazole and the mitotic index was obtained with flow cytometric-based Histone H3 S10p staining. The average percentages of mitotic cells are shown. Error bars represent the standard deviations of at least triplicate samples.
(E) Control and ATM knockdown cells expressing H2B-GFP were imaged at the onset of mitosis to monitor chromosome dynamics. Representative fluorescence video microscopy series are shown. Arrows indicate maloriented and unaligned chromosomes.
(F) The average time from nuclear envelope breakdown (NEB) to anaphase onset in stable cell lines expressing control or ATM shRNA was measured by time-lapse microscopy. Cell numbers were 21 (Control shRNA), 20 (ATM shRNA-1), and 21 (ATM shRNA-2). Error bars represent the standard deviation.
(G) Percentage of aberrant anaphases in ATM-depleted and control cells as shown in Figure 5E. Error bars represent the standard deviations of at least triplicate samples.
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
ATM Ser1403 Phosphorylation Is Critical for Activation of the Spindle Checkpoint
(A) HeLa cells transfected with vector, WT, S1403A, or S1403D ATM were exposed to nocodazole for 16 hr and stained for Histone H3p to obtain the mitotic index. Average percentages of mitotic cells are shown. Error bars represent the standard deviations of at least triplicate samples.
(B) Expressions of flag-tagged ATM proteins were assessed by western blot.
(C) Live cell time-lapse imaging.
(D) Vector, ATM-WT or ATM-S1403A was cotransfected with H2B-GFP into HeLa cells. The time from NEB to anaphase onset was measured by time-lapse microscopy. Each diamond in the scatter plot represents a single cell. Cell numbers were 81 (vector), 66 (ATM-WT), and 66 (ATM-S1403A).
(E and F) The EBV-transformed lymphoblast cell line, GM1526 (E), and the SV-40 transformed fibroblast cell line, GM9607 (F), were transfected with vector, WT, or S1403A ATM by electroporation. Thirty-six hours after transfection, cells were treated with mock or nocodazole followed by flow cytometry-based assay to assess the mitotic index. Expression of transfected proteins is also shown. Error bars represent the standard deviations of at least triplicate samples.
(G) Vector, WT, or S1403A ATM was cotransfected with H2B-GFP into GM9607 cells. The time from NEB to anaphase onset was measured by time-lapse microscopy. Cell numbers were 49 (vector), 51 (ATM-WT), and 52 (ATM-S1403A).
(H) GM9607 cells were transfected with vector, WT, S1981A, or S1403A ATM. Thirty-six hours after transfection, cells were treated with mock or IR (6Gy), and 2 hr later cells were subjected to H3 Ser10p staining by flow cytometry. Average percentages of mitotic cells are shown. Error bars represent the standard deviations of at least triplicate samples.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
ATM Phosphorylates Bub1 on Ser314 to Activate the Spindle Checkpoint
(A and B) Isogenic HeLa cell lines with proficient (control shRNA) or deficient (ATM shRNA) ATM were treated with mock or nocodazole and subjected to immunoblotting with the indicated antibodies.
(C) HeLa cells transfected with vector, WT, or S1403A ATM were treated with nocodazole and immunoblotted with the indicated antibodies.
(D) HeLa cells were transfected with vector, WT, or S314A Bub1. Thirty-six hours after transfection, cells were treated with nocodazole and subjected to flow cytometry analysis. The averages of the mitotic index of at least three independent samples are shown. Error bars represent the standard deviation.
Molecular Cell  , DOI: ( /j.molcel )
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