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Figure Lengend Snippet: Identification of TPX2/Aurora A as 53BP1 effectors. (A) Flag-HA-53BP1 and associated proteins were isolated from HeLa-S nuclear extract using sequential Flag and HA immunoaffinity purification. The final eluted material was analyzed by SDS-PAGE and silver staining. (B) The 53BP1 complex from A was analyzed by MS/MS twice, and the total/unique peptide numbers for each protein are shown for each run. (C) Nuclear extracts from HeLa-S cells were immunoprecipitated with control IgG or TPX2 antibody and Western blotted as shown. (D) GST, or the indicated GST-53BP1 fragments, were immobilized on glutathione-Sepharose, and binding with full-length recombinant His-TPX2 was tested. Bound material was analyzed by Western blot or Coomassie Blue staining. (E) Schematic of 53BP1 and summary of TPX2 binding data. (F) GST, GST-TPX2, or the indicated GST-TPX2 fragments were immobilized on glutathione-Sepharose and assessed for binding to full-length Flag-53BP1, as in D. (G) Schematic of TPX2 and summary of 53BP1 binding data. (H) GST, and increasing equimolar amounts of GST-TPX2 or GST- TPX2-Δ150-394, were tested for 53BP1 binding as done in F. (I) Recombinant His-Aurora A was immobilized on Ni-NTA and incubated with GST, GST-TPX2, or GST- TPX2-Δ150-394. Bound material was washed, subsequently incubated with Flag-53BP1, washed again, and analyzed for binding by Western blot or Coomassie Blue staining.

Article Snippet: Primary antibodies used were rabbit anti-TPX2 (1:500, Bethyl; A300-429A) and mouse anti–Aurora A (1:200; Abcam; 13824).

Techniques: Isolation, Immunoaffinity Purification, SDS Page, Silver Staining, Tandem Mass Spectroscopy, Immunoprecipitation, Western Blot, Binding Assay, Recombinant, Staining, Incubation

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Figure Lengend Snippet: TPX2 and Aurora A regulate BRCA1 recruitment and function. (A) U2OS cells expressing the indicated shRNAs were irradiated (5 Gy) and analyzed for 53BP1 and pH2A.X foci formation. Percentage of cells with ≥10 53BP1 foci ± SD is shown on the right. Scale bar, 10 µm. Foci were quantitated from three biological replicates with n ≥ 200 cells per experiment. Statistics: Student’s two-tailed t test; *, P < 0.05. (B) U2OS cells expressing the indicated shRNAs were treated with camptothecin (CPT) as shown, and chromatin-bound RPA was quantified by flow cytometry. Bar graph indicates the percentage of S/G2 cells that are RPA positive, as determined by 7-AAD staining from four biological replicates. Error bars represent ± SD. Statistics: Student’s two-tailed t test; *, P < 0.05. (C) Quantitation of HR efficiency of DR-GFP U2OS cells treated with the indicated shRNA and with or without the Flag-ISce expression vector. Cells were analyzed for GFP positivity via flow cytometry in three biological replicates as described in Materials and methods. Error bars indicate ± SD. Statistics: Student’s two-tailed t test; *, P < 0.05. (D and E) U2OS cells expressing the indicated shRNAs were irradiated (5 Gy) and analyzed for BRCA1 and p.H2A.X foci formation. Percentage of cells with ≥10 BRCA1 foci ± SD is shown on the right. Scale bar, 10 µm. Foci were quantitated from three biological replicates with n ≥ 250 cells per experiment. Statistics: Student’s two-tailed t test; *, P < 0.05. (F) Schematic of FUCCI cell cycle indicator expression pattern of mK02-Cdt (red; G1) and mAG-Geminin (green; S/G2/M; left). U2OS-FUCCI cells were treated with the indicated shRNAs, and S/G2-phase cells (yellow or green cells; indicated by white arrows) were analyzed for BRCA1 foci formation (right). Percentage of S/G2 cells with ≥10 BRCA1 foci ± SD is shown on the right. Scale bar, 10 µm. Foci were quantitated from three biological replicates with n ≥ 250 cells per experiment. Statistics: Student’s two-tailed t test; *, P < 0.05. (G and H) U2OS cells expressing the indicated shRNAs were irradiated (5 Gy) and analyzed for Rad51 foci. Percentage of cells with four or greater Rad51 foci ± SD is shown on the right. Scale bar, 10 µm. Foci were quantitated from three biological replicates with n ≥ 250 cells per experiment. Statistics: Student’s two-tailed t test; *, P < 0.05.

Article Snippet: Primary antibodies used were rabbit anti-TPX2 (1:500, Bethyl; A300-429A) and mouse anti–Aurora A (1:200; Abcam; 13824).

Techniques: Expressing, Irradiation, Two Tailed Test, Flow Cytometry, Staining, Quantitation Assay, shRNA, Plasmid Preparation

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Figure Lengend Snippet: TPX2 and Aurora A promote replication fork stability. (A) Representative DNA fiber images of control, TPX2-depleted, and Aurora A–depleted U2OS cells treated with HU (4 mM) for 120 min. IdU, red; CldU, green. Scale bar, 50 µm. (B) Schematic of single-molecule DNA fiber track analysis (top) and size distribution of IdU track length in TPX2- and Aurora A–depleted cells in the presence and absence of HU or Mirin (bottom). Mirin (50 µM) was added concomitantly with HU treatment, as indicated. Out of two biological replicates; n ≥ 300 tracks were scored for each dataset. Bars represent median. Statistics: Mann–Whitney; ****, P < 0.0001; n.s., not significant. (C) Labeling and HU treatment schematic (top) and size distribution of IdU track length in U2OS cells expressing the indicated combination of shRNA and TPX2 expression vector as performed in B (bottom). (D) Size distribution of IdU track length in U2OS cells expressing the indicated combination of shRNA and Aurora A expression vector as performed in B. (E) U2OS cells were pulse labeled with 10 µM EdU and subjected to a 1-h thymidine chase (10 µM). Proteins bound to nascent DNA were purified by aniPOND as described in Materials and methods and analyzed by Western blot. (F) Size distribution of IdU track length in BRCA1-deficient (UWB1) and -proficient (UWB1 + BRCA1) cells in the presence of HU. Cells were infected with control or TPX2 shRNA before IdU and CldU labeling, as indicated. Fiber analysis was performed as in B.

Article Snippet: Primary antibodies used were rabbit anti-TPX2 (1:500, Bethyl; A300-429A) and mouse anti–Aurora A (1:200; Abcam; 13824).

Techniques: MANN-WHITNEY, Labeling, Expressing, shRNA, Plasmid Preparation, Purification, Western Blot, Infection

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Figure Lengend Snippet: TPX2/Aurora A negatively regulate 53BP1 in the DNA damage response. (A) WT U2OS or 53BP1 knockout cells expressing the indicated shRNAs were irradiated (5 Gy) and analyzed for BRCA1 and pH2A.X foci formation. (B) U2OS cells treated as in A were also analyzed by Rad51 and pH2A.X foci. Scale bars, 10 µm. (C) Quantitation of BRCA1 foci from A. Foci were quantitated from three biological replicates with n ≥ 200 cells per experiment. Error bars indicate ±SD. Statistics: Student’s two-tailed t test; *, P < 0.05. (D) Quantitation of Rad51 foci from B. Foci were quantitated from three technical replicates with n ≥ 200 cells per experiment. Error bars indicate ±SD. Statistics: Student’s two-tailed t test; *, P < 0.05. (E) Size distribution of IdU track length in U2OS cells. WT, 53BP1 KO, or PTIP KO cells were treated with control or TPX2 shRNA before IdU and CldU labeling, as indicated. Out of two biological replicates; n ≥ 300 tracks scored for each dataset. Bars represent median. Statistics: Mann–Whitney; ****, P < 0.0001; n.s., not significant. (F) Size distribution of IdU track length in MEFs. WT or 53BP1 −/− cells were treated with control or TPX2 shRNA before labeling and HU treatment as indicated. Fiber analysis was performed as in E. (G) Size distribution of IdU track length in U2OS cells expressing the indicated combination of shRNA and TPX2 expression vector. Out of three biological replicates; n ≥ 300 tracks scored for each dataset. Bars represent median. Statistics: Mann–Whitney; ****, P < 0.0001; n.s., not significant. (H) U2OS WT or 53BP1-knockout cells were treated with the indicated shRNAs and subsequently treated with HU, as described in Materials and methods. Cell viability after 48 h of drug exposure was measured by MTS assay. Graph is a representative of three biological replicate experiments. Error bars indicate ±SD with n = 5 technical replicates.

Article Snippet: Primary antibodies used were rabbit anti-TPX2 (1:500, Bethyl; A300-429A) and mouse anti–Aurora A (1:200; Abcam; 13824).

Techniques: Knock-Out, Expressing, Irradiation, Quantitation Assay, Two Tailed Test, shRNA, Labeling, MANN-WHITNEY, Plasmid Preparation, MTS Assay

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Figure Lengend Snippet: TPX2/Aurora A regulate DSB repair and replication fork stability via negative regulation of 53BP1. (A) Model of TPX2/Aurora A regulation of 53BP1 in response to double-strand breaks. (B) Model of TPX2/Aurora A function in response to replication stress. See text for details.

Article Snippet: Primary antibodies used were rabbit anti-TPX2 (1:500, Bethyl; A300-429A) and mouse anti–Aurora A (1:200; Abcam; 13824).

Techniques: