Heart tissues were dissected and fixed in 4% paraformaldehyde at 4°C overnight. The fixed tissues were washed three times with PBS and equilibrated in 30% sucrose at 4°C for 2 days before freezing and cryosectioning. Eight micrometer frozen sections were blocked at 2% goat serum and then stained with the respective primary antibodies at 10 ug/ml at 4˚C overnight. Anti-mouse primary antibodies used: COLA1 (abcam, ab34710), cTnT (abcam, ab8295), Ki67 (eBiosciences 14-5698-82), pH3 (Millipore, 06-570) and Aurora B (abcam, ab2254). Anti-human primary antibodies used: cTnT (RnD systems, MAB1874), Ki67 (abcam, ab15580) and Aurora B (abcam, ab2254). Alexa-Fluor-488- or Alexa-Fluor-546-conjugated secondary antibodies (Invitrogen) were used at room temperature for 30 minutes in the dark. Slides were mounted with DAPI-containing fluorescence mounting medium (Dako) and fluorescence was detected with an upright fluorescence microscope, inverted fluorescence microscope or confocal microscope (all Leica). Images were processed with the ImageJ software and cTNT coverage was analyzed based on this formula: cTnT+ area/total area. For blind cell count of proliferating cardiomyocytes, contaminating cell types without cTnT expression and background staining without DAPI+ nuclei were excluded.
Aurora b
Manufactured by Abcam
Sourced in United States, United Kingdom
Aurora B is a protein kinase that plays a crucial role in chromosome segregation and cytokinesis during cell division. It is an essential component of the chromosomal passenger complex, which regulates the alignment and separation of chromosomes during mitosis.
Automatically generated - may contain errors
Lab products found in correlation
Securin, by Abcam (3 mentions)
P plk1 thr210, by Cell Signaling Technology (2 mentions)
Poly hydroxyethylmethacrylate polymer poly hema, by Santa Cruz Biotechnology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Separase, by Abcam (2 mentions)
Aurora a, by Leica (2 mentions)
Cyclin e, by Thermo Fisher Scientific (2 mentions)
β actin, by Abcam (2 mentions)
Cyclin b1, by Thermo Fisher Scientific (2 mentions)
Cyclin a, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (2 mentions)
Lamin b, by Proteintech (2 mentions)
Poly l lysine coated coverslips, by Corning (2 mentions)
Inverted fluorescence microscope, by Leica (1 mentions)
Confocal microscope, by Leica (1 mentions)
Alexa fluor 488 or alexa fluor 546 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Upright fluorescence microscope, by Leica (1 mentions)
Dapi containing fluorescence mounting medium, by Agilent Technologies (1 mentions)
Cola1, by Abcam (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
18 protocols using aurora b
1
Immunostaining and Quantification of Cardiac Tissue
2
Western Blot Analysis of CHMP4C and Cell Signaling
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The cells were harvested and lysed in RIPA lysis buffer (Thermo Scientific Pierce, Waltham, MA, USA). The protein was collected at 12,000× g for 15 min at 4 °C and measured by BCA protein assay kit (Thermo Scientific Pierce). Equal amounts of protein were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and blotted on nitrocellulose membranes for Western blot analysis. The membranes were blocked in 5% nonfat milk and then incubated with the following primary antibodies: CHMP4C (Abcam, Cambridge, UK), phosphorylated (p)—CHMP4C (Abmart, Arlington, MA, USA), Aurora B (Abcam), p53 (Cell Signaling Technology, Boston, MA, USA), p21 (Cell Signaling Technology) and β-actin (Cell Signaling Technology). The CHMP4C antibody is diluted in 1:500, and the rest were used in 1:1000 dilutions. Membranes were washed in tris-buffered saline containing 0.5% tween-20 and then incubated with goat anti-rabbit lgG (Abcam, 1:2000) or goat anti-mouse lgG (Abcam, 1:3000) conjugated to horseradish peroxidase for 1 h at room temperature. The membranes were detected using Chemiluminescence liquid (Thermo Scientific Pierce) according to the manufacturer’s protocol and analyzed by the Image J software (Bio-Rad, Hercules, CA, USA).
3
Immunostaining of Paraffin-Embedded Tissues
4
Western Blot Analysis of Cellular Proteins
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Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) and incubated overnight at 4°C with antibody against Rab18 (1:1,000; Proteintech), p-Rb, survivin, cleaved caspase 3, cleaved PARP, cytochrome c (1:1,000; Cell Signaling Technology), Aurora B, Aurora B (phospho S227) (1:800; Abcam, Cambridge, UK), and GAPDH (1:3,000; Santa Cruz Biotechnology). After incubation with HRP-linked anti-mouse/rabbit IgG (1:3,000; Santa Cruz Biotechnology) at 37°C for 2 hours, proteins were visualized using enhanced chemiluminescence system (Thermo Fisher Scientific, Waltham, MA, USA) and DNR gel documentation system (DNR BioImaging Systems, Neve Yamin, Israel).
5
Assessing hiPSC-CM Proliferation Post-Differentiation
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After four weeks of the initiation of cardiomyogenic differentiation, hiPSC-CM was stained for the presence of human cardiac Troponin T (hcTnT) and other cell cycle proliferation markers. Nuclei were identified with DAPI staining. Cell proliferation was evaluated via immunofluorescence imaging and labeling of Ki67 (EMD MilliporeSigma, MAB4190, Rockville, MD, USA) expression and quantified as a percentage of the positively stained cardiomyocytes. Cells in S-phase of cell cycle were identified via analyses of Bromodeoxyuridine (BrdU; Abcam, #ab6326, Boston, MA, USA) incorporation and quantified as a percentage of the positively stained cardiomyocytes. Similarly, cells undergoing cytokinesis were identified via Aurora B (Abcam, #ab3609, Boston, MA, USA) expression and quantified as a percentage of the positively stained cardiomyocytes. Likewise, cells in M-phase of cell cycle were identified via analyses of phosphorylated histone H3 (PH3; EMD Millipore Sigma, #06-570, Rockville, MD, USA) expression and quantified as the percentage of positively stained cardiomyocytes.
6
Antibody Procurement and Chemical Reagent Acquisition
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Antibodies were obtained as follows: p-PLK1 (Thr210) (Cell Signalling Technology); Aurora A (Millipore); Aurora B (Abcam); PLK1 (clone F-8), VCAM-1 (clone H-276), ICAM-1 (clone H-108), Bcl-2 (clone N-19), Bax (clone N-20), survivin (clone FL-142), p21 (clone C-19), cyclin B1 (clone GNS1), Wee1 (clone C-20), CDC25 (clone C-20), c-Myc (clone 9E10) and β-actin (Santa Cruz Biotechnology).
Tivozanib was purchased from AdooQ BioScience (Irvine, CA, USA). Gefitinib (EGFR small molecule inhibitor) and temozolomide (a DNA alkylating agent) were obtained from ChemieTek (Indianapolis, IN, USA). Irinotecan (a topoisomerase I inhibitor) was purchased from pharmacy of Shariati hospital (Tehran, Iran). Poly-hydroxyethylmethacrylate polymer (poly-HEMA) was obtained from Santa Cruz Biotechnology.
Tivozanib was purchased from AdooQ BioScience (Irvine, CA, USA). Gefitinib (EGFR small molecule inhibitor) and temozolomide (a DNA alkylating agent) were obtained from ChemieTek (Indianapolis, IN, USA). Irinotecan (a topoisomerase I inhibitor) was purchased from pharmacy of Shariati hospital (Tehran, Iran). Poly-hydroxyethylmethacrylate polymer (poly-HEMA) was obtained from Santa Cruz Biotechnology.
7
Antibody Production and Validation
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The anti-DTL antibody was produced using GST–L2DTL protein, as previously described.9 (link) The following antibodies were used: cyclin A, cyclin B1, cyclin E, CDK1, and CDK2 (Thermo Fisher Scientific); β-actin, separase, securin, and Aurora B (Abcam, Cambridge, UK); TPX2 (Sigma-Aldrich Co., St Louis, MO, USA); Aurora A (Leica, Milton Keynes, UK); and NDC80 (Proteintech, Rosemont, IL, USA).
8
Antibody Profiling of Cell Cycle Regulators
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The following antibodies were used for immunoprecipitation (IP) and immunoblotting (IB, dilutions shown): NudC (70/1, rabbit, 1:1000) [32 (link)], NudC (G1, goat, 1:1000) [33 (link)], NudC (2D9, mouse, 1:2000) [32 (link)], Aurora B (BD, mouse, 1:800), Aurora B (Abcam, rabbit, 1:2000), α-tubulin (GeneTex, rabbit, 1:2000), α-tubulin (Sigma, mouse, 1:1000), β-tubulin(tub2.1) (Sigma, mouse, 1:1000), and FLAG (Sigma, mouse, 1 μl/mg protein for IP). The following antibodies were used for immunofluorescence (1:1000, unless otherwise indicated): NudC (G1, goat) [33 (link)], pSerNudC (R2, rabbit) [32 (link)], Borealin (MBL International, mouse), PRC1 (Abcam, rabbit), MKLP-1 (Cell Signaling, rabbit), and pTSS-INCENP834-902 (gift of Dr. Michael Lampson, University of Pennsylvania; rabbit) [36 (link)], Spc25 (gift of Dr. P. Todd Stukenberg, University of Virginia Medical Center; rabbit; 1:700) [37 (link)], and CREST-SH autoserum (gift of Dr. Bill R. Brinkley, Baylor College of Medicine; human; 1:10,000) [38 (link)]. Protease inhibitors and nocodazole, the microtubule depolymerizing agent, were purchased from Sigma. ZM447439, an Aurora B inhibitor, was purchased from Tocaris and used at a final concentration of 2 μM.
9
Molecular Signaling Pathways in Cancer
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TAK901 (#T2709) was purchased from TargetMol (Shanghai, China). Primary antibodies were as follows: SREBP-1 (2A4) (#sc-13551, Santa Cruz Biotechnology, Santa Cruz, CA, USA), SREBP-2 (1C6) (#sc-13552, Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-GSK-3β (Ser9) (D85E12) (#5558, CST, Danvers, MA, USA), β-actin (#20536-1-AP, Proteintech, Wuhan, China), Ki67 (Servicebio, Wuhan, China),histone H3 (#4499s, CST, Danvers, MA, USA), phospho-histone H3 (Ser10) (#53348T, CST, Danvers, MA, USA), Aurora A (#45-8900, Invitrogen, Carlsbad, CA, USA), Aurora B (#ab2254, abcam, Waltham, MA, USA) and c-Myc (#9402S, CST, Danvers, MA, USA). Secondary antibodies were as follows: anti-mouse IgG, HRP-linked antibody (#7076, CST, Danvers, MA, USA), anti-rabbit IgG, HRP-linked antibody (#7074, CST, Danvers, MA, USA).
10
Antibody Reagents and Cell Treatment Protocols
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Antibodies were obtained as follows: Aurora B (Abcam); EGFR, HER2, HER3 (clone 2F12) and HER3-neutralising monoclonal antibody (clone H3.105.5) (Millipore); p-EGFR (Tyr1068), p-HER2 (Tyr1248), p-HER3 (Tyr1289; clone 21D3), p-HER4 (Tyr1284; clone 21A9) and p-PLK1 (Thr210) (Cell Signalling Technology); HER4 (clone C-7), PLK1 (clone F-8), FOXM1 (clone C-20), survivin (clone FL-142) and β-actin (Santa Cruz Biotechnology). Dacomitinib and BI 2536 (a specific inhibitor of polo-like kinase 1) were purchased from Adooq Bioscience (Irvine, CA, USA) and were dissolved in DMSO. The final concentrations of DMSO did not exceed than 0.1% [v/v] in all the treatments. Erlotinib (EGFR small molecule inhibitor) was obtained from Chemietek (Indianapolis, IN, USA). Cetuximab (a ligand-blocking anti-EGFR mAb), trastuzumab (anti-HER2 mAb), cisplatin and doxorubicin (DNA-damaging drugs), paclitaxel (a taxane inhibitor of microtubule disassembly), vincristine (a mitosis-blocking agent), carboplatin (an alkylating agent) and gemcitabine (a nucleoside analogue which inhibits DNA synthesis) were purchased from the pharmacy of Shariati hospital (Tehran, Iran). Poly-hydroxyethylmethacrylate polymer (poly-HEMA) was obtained from Santa Cruz Biotechnology. Recombinant HRGβ-1 was purchased from Peprotech.
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