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: Oncotarget

Article Title: Heterogeneity in sarcoma cell lines reveals enhanced motility of tetraploid versus diploid cells

doi: 10.18632/oncotarget.14291

Figure Lengend Snippet: CINSARC genes RNA Seq data for MFH152 diploid and tetraploid clones normalized to IMR90 expression levels

Article Snippet: Polyclonal antibodies against PRC1 (Santa Cruz), Tpx2 [ ]; Kif23 [ ] were used.

Techniques: RNA Sequencing, Clone Assay, Expressing

Journal: Cancer Research

Article Title: Proteomic Characterization of the Angiogenesis Inhibitor SU6668 Reveals Multiple Impacts on Cellular Kinase Signaling

doi: 10.1158/0008-5472.can-05-0574

Figure Lengend Snippet: Figure 2. SU6668 inhibits Aurora kinase activity and cell cycle progression in human HeLa cells. A, distribution of the DNA content in control and SU6668-treated cells. HeLa cells were incubated with different concentrations of SU6668 or DMSO as a control for 15 hours and then stained with propidium iodide for flow cytometry analysis. Where indicated, inhibitor-treated cells were washed and then cultured for a further 4 hours in fresh medium without SU6668 (wash-out). B, after 15 hours of treatment with 20 Amol/L SU6668 or DMSO as a control, HeLa cells were subjected to immunofluorescence analysis using the indicated antibodies (bar, 10 Am; Overlays: blue, DNA; red, histone H3 phosphorylated on Ser10; green, TPX2). C, after 15 hours of treatment with 20 Amol/L, SU6668 cells were released into fresh medium without SU6668 for 30 minutes prior to immunofluorescence analysis as described in (B). D, HeLa cells treated with SU6668 as described in (B) and then stained with antibodies specific for a-tubulin and Aurora A.

Article Snippet: The following antibodies were used for immunofluorescence: against human full-length TPX2 protein (1:10,000; ref. 19), against a-tubulin (Sigma), against histone H3 phosphorylated on Ser10 (Cell Signaling Technology, Beverly, MA) and against Aurora A (Abcam, Cambridge, MA, all used at 1:500).

Techniques: Activity Assay, Control, Incubation, Staining, Flow Cytometry, Cell Culture, Immunofluorescence

Journal: Oncology letters

Article Title: Reversal of the tamoxifen‑resistant breast cancer malignant phenotype by proliferation inhibition with bromosulfonamidine amino‑podophyllotoxin.

doi: 10.3892/ol.2024.14506

Figure Lengend Snippet: Figure 4. Effect of BSAPPT on apoptosis and cycle‑related gene or protein expression in MCF‑7, MCF7/TAMR and other cancer cells. (A) mRNA expres‑ sion levels of genes linked to the cell cycle and apoptosis were measured by qPCR both before and after MCF‑7 and MCF7/TAMR cells were treated with 10 µg/ml BSAPPT. (B) Western blotting detection of apoptotic and cycle‑related protein expression variations in MCF‑7 and MCF7/TAMR cells before and after using 10 µg/ml BSAPPT. Results of qPCR analysis that assessed differences in the level of expression of genes linked to the cell cycle and apoptosis before and after (C) A549 and (D) MDA‑MB‑231 cells were treated with 10 µg/ml BSAPPT. *P<0.05; **P<0.01; ***P<0.001. BSAPPT, bromosulfonamidine amino‑podophyllotoxin; qPCR, quantitative PCR; Bcl‑2, B‑cell lymphoma 2; Caspase, cysteine aspartic acid‑specific protease; PLK, polo like kinase; CCNB1, cyclin B1; TPX2, targeting protein for Xklp2; Bax, Bcl‑2 associated X; Cyt‑C, cytochrome c; Apaf‑1, apoptotic protease activating factor 1.

Article Snippet: Mouse anti‐human Caspase‐9 antibodies (cat. no. 9508S; 1:1,000) were purchased from Cell Signaling Technology, Inc., rabbit anti‐human Bcl‐2 (cat. no. BA0412; 1:1,000) and cyclin B1 (CCNB1; cat. no. BA0766; 1:1,000) antibodies were purchased from Wuhan Boster Biological Technology, Ltd., rabbit anti‐human polo like kinase (PLK)‐1 antibodies (cat. no. 10305‐1‐AP; 1:1,000) and targeting protein for Xklp2 (TPX2) antibodies (cat. no. 11741‐1‐AP; 1:2,000) were purchased from Proteintech Group, Inc., and the rabbit anti‐human PLK‐4 antibodies (cat. no. A9863; 1:1,000) were purchased from ABclonal Biotech Co., Ltd.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction

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