Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Combined functional genome survey of therapeutic targets for hepatocellular carcinoma.

doi: 10.1158/1078-0432.CCR-09-2214

Figure Lengend Snippet: Fig. 5. Protein expression in HCC. Hematoxylin and eosin (HE) staining (original magnification, × 100) and immunoperoxidase staining (original magnifications, × 100 and × 400) of AKR1B10, HCAP-G, RRM2, and TPX2 proteins in HCC and adjacent nontumorous liver tissue. The specificity of antibodies was determined by immunoblotting of the KIM-1 cell lysate (left). N, nontumorous liver.

Article Snippet: Anti-TPX2 antibody was purchased from Novus Biologicals.

Techniques: Expressing, Staining, Immunoperoxidase Staining, Western Blot

Journal: Journal of Biological Chemistry

Article Title: TPX2 Inhibits Eg5 by Interactions with Both Motor and Microtubule

doi: 10.1074/jbc.m114.612903

Figure Lengend Snippet: FIGURE 1. Binding of TPX2 and TPX2-710 to microtubules. A, schematic diagram of the TPX2 constructs (left) and Coomassie Brilliant Blue-stained gel of the purified proteins (right). B, co-sedimentation of TPX2 with microtubules. S, supernatant; P, pellet. The concentration of microtubules in each pair of lanes is noted above. Western blots were stained for TPX2 or tubulin. C, quantification of apparent affinity was performed using a quadratic fit. The experiment was performed twice, and the values were averaged. Error bars, S.D.

Article Snippet: The proteins were then transferred to a PVDF membrane and probed using antibodies against TPX2 (Novus Biologicals, Littleton, CO) and tubulin (DM1A, SigmaAldrich).

Techniques: Binding Assay, Construct, Staining, Purification, Sedimentation, Concentration Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: TPX2 Inhibits Eg5 by Interactions with Both Motor and Microtubule

doi: 10.1074/jbc.m114.612903

Figure Lengend Snippet: FIGURE 2. Binding Dynamics of TPX2 and TPX2-710. A, box plot showing release of TPX2 and TPX2–710 from microtubules in the presence of the indicated concentration of KCl added to the buffer. TPX2 fluorescence is reported as arbitrary units (A.U.). Whiskers define the range, boxes encompass the 25th to 75th quartiles, and lines depict the medians. B, TPX2 and TPX2-710 binding to untreated and subtilisin A-digested microtubules; top panels, fluorescence images of TPX2-Halo or TPX2–710-Halo bound to untreated and subtilisin A-digested microtubules; middle, quantification of TPX2 fluorescence; bottom, polyacrylamide gel showing digested and control microtubules. TPX2 fluorescence was measured for at least 60 microtubules for each of two independent experiments; error bars, S.D. C, kymograph of TPX2-Halo and TPX2-710-Halo on microtubules. Vertical scale bar (time), 60 s; horizontal scale bar, 2 m.

Article Snippet: The proteins were then transferred to a PVDF membrane and probed using antibodies against TPX2 (Novus Biologicals, Littleton, CO) and tubulin (DM1A, SigmaAldrich).

Techniques: Binding Assay, Concentration Assay, Fluorescence, Control

Journal: Journal of Biological Chemistry

Article Title: TPX2 Inhibits Eg5 by Interactions with Both Motor and Microtubule

doi: 10.1074/jbc.m114.612903

Figure Lengend Snippet: FIGURE 4. Inhibition of Eg5 by TPX2 requires both binding to the microtubule and an interaction between TPX2 and Eg5. A, kymographs of Eg5-EGFP before andfollowingtheadditionofTPX2orTPX2–710;arrowhead,timeoftheTPX2addition.B,quantificationofEg5-EGFPvelocity;errorbars,S.D.C,kymographofkinesin-1 EGFP dimers walking on microtubules before and after the addition of TPX2 (arrowhead). 1 nM kinesin-1 EGFP (green) and 500 nM TPX2-Halo (red) were used. D, kymographs of Eg5-EGFP (green) before and following the addition of 20 nM TPX2-Halo (red). Right panels, enlarged view. E, kymographs of Eg5-EGFP that was premixedwithTPX2-HaloorTPX2–710-Halo.F,quantificationofEg5-EGFPvelocityinthepresenceof50nMTPX2thatwasHalo-tagged(left)oruntagged(right).Error bars, S.E. Horizontal scale bars (A, C, and E), 1 m; horizontal scale bar (D), 2 m; vertical scale bar, 60 s (A, D, and E) and 5 s (C).

Article Snippet: The proteins were then transferred to a PVDF membrane and probed using antibodies against TPX2 (Novus Biologicals, Littleton, CO) and tubulin (DM1A, SigmaAldrich).

Techniques: Inhibition, Binding Assay

Journal: Asian Pacific journal of tropical medicine

Article Title: TPX2 promotes migration and invasion of human breast cancer cells.

doi: 10.1016/j.apjtm.2015.11.007

Figure Lengend Snippet: Figure 1. mRNA and protein expressions of TPX2 in breast cancer tissue and cell lines. A: Relative TPX2 mRNA expression in breast cancer tissue and tumor-adjacent tissue by RT-PCR; B: Relative TPX2 mRNA expression in different breast cancer cell lines by RT-PCR; C: Relative TPX2 protein expression in breast cancer tissue and tumor-adjacent tissue by Western blot; D: Relative TPX2 protein expression in different breast cancer cell lines by Western blot; NT: Tumor-adjacent tissue; T: Breast cancer tissue; *: P < 0.05.

Article Snippet: The siRNA specific to TPX2 and control RNA were purchased from Santa Cruz Biotechnology (USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Asian Pacific journal of tropical medicine

Article Title: TPX2 promotes migration and invasion of human breast cancer cells.

doi: 10.1016/j.apjtm.2015.11.007

Figure Lengend Snippet: Figure 2. Downregulation in expression of TPX2 in MDA-MB-231 and MCF7 cells by specific TPX2 siRNA. A: Interference effect of siRNA on TPX2 in MDA-MB-231 and MCF7 cells by RT-PCR; B: Interference effect of siRNA on TPX2 in MDA-MB-231 and MCF7 cells by Western blot; **: P < 0.05.

Article Snippet: The siRNA specific to TPX2 and control RNA were purchased from Santa Cruz Biotechnology (USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Asian Pacific journal of tropical medicine

Article Title: TPX2 promotes migration and invasion of human breast cancer cells.

doi: 10.1016/j.apjtm.2015.11.007

Figure Lengend Snippet: Figure 3. Effect of TPX2 downregulated by siRNA on proliferation of breast cancer cells. A: Effects of downregulated TPX2 and control siRNA on proliferation of MDA-MB-231 cell by MTT assay; B: Effects of downregulated TPX2 and control siRNA on proliferation of MCF7 cell by MTT assay; *: P < 0.05.

Article Snippet: The siRNA specific to TPX2 and control RNA were purchased from Santa Cruz Biotechnology (USA).

Techniques: Control, MTT Assay

Journal: Asian Pacific journal of tropical medicine

Article Title: TPX2 promotes migration and invasion of human breast cancer cells.

doi: 10.1016/j.apjtm.2015.11.007

Figure Lengend Snippet: Figure 4. Effect of TPX2 downregulated by siRNA on breast cancer cell migration by Transwell assay. A: MDA-MB-231 cell; B: MCF7 cell; *: P < 0.05.

Article Snippet: The siRNA specific to TPX2 and control RNA were purchased from Santa Cruz Biotechnology (USA).

Techniques: Migration, Transwell Assay

Journal: Asian Pacific journal of tropical medicine

Article Title: TPX2 promotes migration and invasion of human breast cancer cells.

doi: 10.1016/j.apjtm.2015.11.007

Figure Lengend Snippet: Figure 5. Effect of TPX2 downregulated by siRNA on breast cancer cell invasion by Transwell assay. A: MDA-MB-231 cell; B: MCF7 cell; *: P < 0.05.

Article Snippet: The siRNA specific to TPX2 and control RNA were purchased from Santa Cruz Biotechnology (USA).

Techniques: Transwell Assay

Journal: bioRxiv

Article Title: AURKA destruction is decoupled from its activity at mitotic exit but suppresses interphase activity

doi: 10.1101/850917

Figure Lengend Snippet: A pT288 antibody detects active AURKA only in mitotic cells. Cells were synchronized as described in Materials and Methods and blotted for pT288, total AURKA and other mitotic markers. B pT288 signal is sensitive to AURKA-specific inhibitor MLN8237 by IF on mitotic cells from a MeOH-fixed unsynchronized population (upper panel) or by immunoblot of STLC-arrested mitotic cells treated for 3 hours at the indicated doses (lower panel). AURKA-specific pT288 signal is restricted to centrosomes and spindle pole bodies (marked by γ-tubulin, TUBG1). Bars, 10 μm. See also Figure S1. C-E Quantification of pT288-AURKA during mitotic exit. C, D Unsynchronized cell populations were fixed and stained as in B . Cells were judged to be at different stages of mitosis according to DAPI staining ( C ) and scored for mean pT288 AURKA signal measured in a fixed ROI centred on TUBG1 signal at centrosomes or spindle poles ( D ). G2 and prophase (P), n=10; prometaphase (PM), n=15; metaphase (M), n=30; anaphase (A), n=30; and telophase (T), n=26. M vs A, not significant (n.s.); A vs T, p < 0.0001 (***), Students’ t-test. E Cells were synchronized in 5 μM STLC and released by checkpoint inhibition using 10 μM AZ3146, with extracts harvested at times indicated. These were examined by immunoblotting for AURKA, pT288-AURKA and TPX2 levels. Disappearance of Cyclin B1 (CCNB1) acts as marker for mitotic exit, level of vinculin (VCL) as loading control.

Article Snippet: Primary antibodies for immunoblot were as follows: AURKA mouse mAb (1:1000; Clone 4/IAK1, BD Transduction Laboratories), phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (1:1000; clone D13A11 XP® Rabbit mAb, Cell Signalling), rabbit polyclonal TPX2 antibody (1:1000; Novus Biological), Cdh1 mouse mAb (1:50; gift from T. Hunt and J. Gannon), CDC20 mouse mAb (1:1000; Santa Cruz sc13162), AURKB rabbit polyclonal antibody (1:1000; Abcam ab2254), mouse monoclonal Cyclin B1 (1:1000; BD 554177), DRP1 rabbit polyclonal (1:500; Bethyl lab), rabbit polyclonal Tubulin (1:2000; Abcam ab6046), mouse mAb anti-Vinculin (1:1000; clone hVIN-1, Sigma-Aldrich), rabbit anti-GFP (1:1000; 11814460001, Roche).

Techniques: Western Blot, Staining, Inhibition, Marker, Control

Journal: bioRxiv

Article Title: AURKA destruction is decoupled from its activity at mitotic exit but suppresses interphase activity

doi: 10.1101/850917

Figure Lengend Snippet: U2OS ( A,B ) and FZR1 KO ( C,D ) cells were transfected with TPX2(1-43)-CFP and synchronized through mitotic exit as described in the legend to . Quantitative immunoblotting of cell lysates shows that loss of pT288-AURKA during mitotic exit is delayed in the presence of TPX2(1-43) in both parental and FZR1 KO cells. Cyclin B1 (CCNB1) is used as marker for mitotic exit, level of vinculin (VCL) as loading control. Bar charts (B, D) show pT288 signal normalized against vinculin. Results presented are mean values from 3 independent experiments ± S.D. E AURKA inactivation is phosphatase dependent. U2OS cells undergoing mitotic exit were treated with PP1 inhibitor 3nM tautomycin 10 minutes after relief of checkpoint inhibition by AZ3146. Lysates harvested at the indicated time points after AZ3146 treatment were subject to immunoblot analysis.

Article Snippet: Primary antibodies for immunoblot were as follows: AURKA mouse mAb (1:1000; Clone 4/IAK1, BD Transduction Laboratories), phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (1:1000; clone D13A11 XP® Rabbit mAb, Cell Signalling), rabbit polyclonal TPX2 antibody (1:1000; Novus Biological), Cdh1 mouse mAb (1:50; gift from T. Hunt and J. Gannon), CDC20 mouse mAb (1:1000; Santa Cruz sc13162), AURKB rabbit polyclonal antibody (1:1000; Abcam ab2254), mouse monoclonal Cyclin B1 (1:1000; BD 554177), DRP1 rabbit polyclonal (1:500; Bethyl lab), rabbit polyclonal Tubulin (1:2000; Abcam ab6046), mouse mAb anti-Vinculin (1:1000; clone hVIN-1, Sigma-Aldrich), rabbit anti-GFP (1:1000; 11814460001, Roche).

Techniques: Transfection, Western Blot, Marker, Control, Inhibition

Journal: Cancer Research

Article Title: Tumor Cell Dependence on Ran-GTP–Directed Mitosis

doi: 10.1158/0008-5472.can-07-5279

Figure Lengend Snippet: Figure 3. Aberrant mitotic spindle assembly and cell death induced by Ran targeting in tumor cells. A, siRNA silencing. HeLa cells were transfected with nontargeted (Control) or Ran-directed siRNA and analyzed by Western blotting at the indicated time intervals. B, differential modulation of Ran effector molecules. HeLa cells transfected with Ran-directed siRNA were analyzed by Western blotting at the indicated time intervals. None, nontransfected cells. *, nonspecific. C, immunofluorescence analysis. HeLa cells transfected with control (left) or Ran-directed (right) siRNA were stained for DNA (DAPI) or TPX2, and analyzed by image merging. D, time course of cell death. HeLa cells were transfected with control or Ran-directed siRNA, harvested at the indicated time intervals, and analyzed for DNA content by propidium iodide staining and flow cytometry. The percentages of cells in sub-G1, G1, or G2-M peaks are indicated.

Article Snippet: Antibodies to survivin (Novus Biologicals), Ran (Novus Biologicals, Cell Signaling, Santa Cruz Biotechnology), a-tubulin (Sigma-Aldrich), TPX2 (Novus Biologicals), RCC1 (Santa Cruz Biotechnology), cytochrome c (BD Biosciences-Clontech), Smac (ProSci), Ran-GAP1 (Abcam), X-linked inhibitor of apoptosis (XIAP; BD Biosciences), caspase-3 (Cell Signaling), or h-actin (Sigma-Aldrich) were used.

Techniques: Transfection, Control, Western Blot, Immunofluorescence, Staining, Flow Cytometry