In total, 26 Ae. umbellulata accessions from seeds supplied by the National BioResource Project-Wheat, Japan (https://shigen.nig.ac.jp/wheat/komugi/ ) were propagated from a single plant by self-pollination (Table 1 ). A tetraploid wheat accession T. turgidum ssp. durum cv. Langdon (Ldn) was used as the female parent and crossed with each of the 26 accessions of Ae. umbellulata (Fig 1 ). All 26 synthetic hexaploid wheat lines with the AABBUU genome (ABU hexaploids, F2 generation) were generated by 0.1% colchicine (Wako Pure Chemical Industries, Osaka, Japan) treatment for 5 h at the seedling stage in each F1 triploid hybrid (F1 generation). Thus, the synthetics share the A and B genomes from Ldn and contain the U genome derived from diverse Ae. umbellulata accessions. All synthetics grew normally in a greenhouse at Kobe University (34°43’N, 135°13’E), and none showed hybrid growth abnormalities such as SGA and GCD [29 (link)]. Four lines of synthetic hexaploid wheat with the AABBDD genome (ABD hexaploids), Ldn/KU-2097 (Syn6214), Ldn/IG126387 (Syn6240), Ldn/PI476874 (Syn6256), and Ldn/KU-2069 (Syn6262), were also used in this study. These four ABD hexaploids showed various heading/flowering time, and did not exhibit any growth abnormalities [37 ,39 ]. The ABD hexaploids were grown under the same conditions as the ABU hexaploids.
Colchicine
Manufactured by Fujifilm
Sourced in Japan
Colchicine is a laboratory product manufactured by Fujifilm. It is a chemical compound used in various research and analytical applications. The core function of Colchicine is to serve as a reference standard and tool for research and development purposes. No further details can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Thapsigargin, by Fujifilm (2 mentions)
Ml240, by Merck Group (2 mentions)
Propyzamide reference material, by Fujifilm (2 mentions)
Oryzalin standard, by Fujifilm (2 mentions)
Concanavalin a (cona), by Fujifilm (1 mentions)
Schneider s insect medium, by Thermo Fisher Scientific (1 mentions)
Latrunculin a, by Fujifilm (1 mentions)
Effectene, by Qiagen (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Nocodazole, by Fujifilm (1 mentions)
Rhosin, by Merck Group (1 mentions)
Ml141, by Merck Group (1 mentions)
Nsc23766, by Abcam (1 mentions)
Cytochalasin d, by Fujifilm (1 mentions)
Vincristine, by Cayman Chemical (1 mentions)
Celltiter glo 2, by Promega (1 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions)
Genistein, by Fujifilm (1 mentions)
Sucrose, by Fujifilm (1 mentions)
Sodium azide, by Kanto Chemical (1 mentions)
17 protocols using colchicine
1
Synthetic Hexaploid Wheat Development
2
Culturing and Transfecting Drosophila S2 Cells
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Drosophila S2 cells were cultured in Schneider's Insect Medium (Gibco) supplemented with 10% FCS and antibiotics at 25°C (Schneider, 1972 (link)). pUAST vectors with actin5Ce-Gal4 drivers were cotransfected using Effectene (Qiagen) according to the manufacturer's instructions, and harvested 36-48 h after transfection. For immunofluorescence or time-lapse imaging, cells were replated on coverslips or glass-bottom dishes coated with Concanavalin A (Wako) and were allowed to spread for 1-2 h (Rogers et al., 2002 (link)). For drug treatments, cells were treated with 1 µM Latrunculin A (Wako) or 10 µM Colchicine (Wako) for 1 h before imaging. For immunofluorescence, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS (PBS-T) for 15 min, and blocked with 5% skimmed milk in Tris-buffered saline (TBS). Primary and secondary antibodies were diluted in the blocking solution. After each antibody incubation, the coverslips were washed three times with PBS-T. The cells were mounted in Vectashield mounting medium (Vector Labs).
3
Plaque Assay for Oncolytic Virus
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Cells were plated at 2 × 105/well in 6-well plates. After 36 h, cells were infected with OVV-LG at an MOI of 0.001. After a 1-h incubation, E-MEM containing 0.8% methyl cellulose supplemented with 5% FBS and reagents was added to each well, and plaque sizes were calculated with a BZ-X700 microscope. Reagents used for plaque assays were as follows: nocodazole (Wako, Osaka, Japan), colchicine (Wako), cytochalasin D (Wako), Rhosin (Merck, Boston, MA, USA), ML-141 (Sigma, St. Louis, MO, USA), and NSC23766 (Abcam, Cambridge, UK).
4
Cytotoxicity Assessment of Anticancer Drugs
5
Colon Epithelial Cell Culture and Lentiviral Transduction
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FHC, a human colon epithelial normal cells, were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured according to the recommended procedure by the supplier. HEK293T and CRC cells (Caco-2, COLO205, SW480, CW-2, LoVo, SW48 and HCT116) were obtained as reported previously (Inaguma et al., 2017 (link); Inoue et al., 2020 (link)). CRC cells were maintained in Dulbecco's Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
The lentivirus vectors for PBK and the control LacZ were constructed using the CSII-EF-MCS plasmid, which was kindly provided by Dr. H. Miyoshi (RIKEN BioResource Center, Tsukuba, Japan). The pcDNA3.1 vector (Invitrogen) was used to express PBK, CDH1cyt (CDH1 cytoplasmic domain, R733 to D882) or firefly luciferase (Luc2CP). The CDH1cyt mutants (CDH1cyt/S840,846,847A and CDH1cyt/S840,846,847D) were generated by PCR-based mutagenesis. The pGEX-5X-1 vector was used for the synthesis and purification of glutathione S-transferase (GST)-tagged proteins.
OTS514, a selective PBK inhibitor, was purchased from Selleck Biotech (Tokyo, Japan) and used to treat cells for 24–72 h. Colchicine was obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and added at a concentration of 1 × 10–7 M for 8 h. MG132, a proteasome inhibitor, was from Selleck Biotech (Tokyo, Japan) and applied at 1 µM for 8 h.
The lentivirus vectors for PBK and the control LacZ were constructed using the CSII-EF-MCS plasmid, which was kindly provided by Dr. H. Miyoshi (RIKEN BioResource Center, Tsukuba, Japan). The pcDNA3.1 vector (Invitrogen) was used to express PBK, CDH1cyt (CDH1 cytoplasmic domain, R733 to D882) or firefly luciferase (Luc2CP). The CDH1cyt mutants (CDH1cyt/S840,846,847A and CDH1cyt/S840,846,847D) were generated by PCR-based mutagenesis. The pGEX-5X-1 vector was used for the synthesis and purification of glutathione S-transferase (GST)-tagged proteins.
OTS514, a selective PBK inhibitor, was purchased from Selleck Biotech (Tokyo, Japan) and used to treat cells for 24–72 h. Colchicine was obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and added at a concentration of 1 × 10–7 M for 8 h. MG132, a proteasome inhibitor, was from Selleck Biotech (Tokyo, Japan) and applied at 1 µM for 8 h.
6
Endocytosis Inhibition Assay for Talaporfin
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Cells were suspended in DMEM or IMDM at 1.0 × 106 cells/mL in 1.5 mL microtubes. After the uptake of talaporfin, the following endocytosis inhibitors were added: 2‐deoxyglucose (2‐DG; Sigma‐Aldrich, St Louis, MO) at a concentration of 50 mmol/L), sodium azide (0.1% w/v; Kanto Chemical, Tokyo, Japan), methyl‐β‐cyclodextrin (5 mmol/L; Wako, Osaka, Japan), genistein (0.1 mmol/L; Wako), sucrose (0.45 M; Wako), chlorpromazine hydrochloride (20 mmol/L; Tokyo Chemical, Tokyo, Japan), cytochalasin β (1 mmol/L; Wako) and colchicine (1 mmol/L; Wako). After culture at 37°C for 30 minutes, the cells were chilled on ice for 30 minutes, washed three times with PBS, fixed with 0.5% formalin and analyzed by flow cytometry. For the inhibition of clathrin‐dependent endocytosis, Pitstop 2 (ab120687; Abcam, Cambridge, UK) was added to the serum‐free DMEM or IMDM to a final concentration of 25 μmol/L, and the cells were cultured at 37°C for 1 hour. Talaporfin was subsequently added to the cells to a final concentration of 30 μg/mL and the cells were washed three times with PBS, fixed with 0.5% formalin, and analyzed by flow cytometry.
7
Detailed Reagent Preparation Protocol
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Eagle's minimal essential medium (E-MEM), MEM non-essential amino acids (MEM-NEAA) solution, penicillin–streptomycin solution (PS), Hank's balanced salt solution (HBSS), 0.25% w/v trypsin-1 mmol/L ethylenediamine tetraacetic acid·4 Na solution, ethanol, dimethyl sulfoxide (DMSO), sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, chloroform, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sodium azide, colchicine, cytochalasin B, heparin sodium, and Lucifer yellow CH dilithium salt fluorescent stain (LY) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Fetal bovine serum (FBS) was purchased from Merck (Darmstadt, Germany). Acetonitrile was purchased from Nacalai Tesque Inc. (Tokyo, Japan).
8
Cell Cycle Analysis of ESCs and TESCs
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ESCs and TESCs were incubated with 0.05 μg/mL colchicine (Wako) for 5 h. After trypsinization with PET solution, 1 × 106 cells were washed in 600 μL of ice-cold PBS, fixed in 1.4 mL of ice-cold 100% ethanol, and incubated for 1 h at 4°C. After removing the ethanol by centrifugation, the cells were resuspended in 1 mL PBS with 100 μg/mL RNase A and incubated for 1 h at RT. Next, 40 μL propidium iodide solution (40 μg/mL) was added to the cells, and the mixture was incubated for 5 min at RT and filtered through 40-μm mesh filters (Kyoshin Rikoh Inc., Tokyo, Japan) prior to analysis. Analysis was performed on a BD Accuri C6 Flow Cytometer (BD Becton, Dickinson and Company, NJ, USA).
9
Preparation of Pharmacological Compound Stock Solutions
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BZ was purchased from Selleck Chemicals (Houston, TX, USA). Azithromycin dihydrate (AZM) was purchased from Tokyo Chemical Industry (Tokyo, Japan). PTX, VNR, and colchicine were obtained from Wako Pure Chemical Industries (Osaka, Japan). BZ was dissolved in dimethyl sulfoxide (DMSO) to make stock solution at a concentration of 1 mM. AZM and PTX were dissolved in 95% ethanol at a concentration of 5 mM as stock solutions. VNR was dissolved in distilled water to prepare a stock solution of 1 mM. colchicine was dissolved in 95% ethanol at a concentration of 10 mM as stock solution.
10
Cytotoxicity Screening of Drug Candidates
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