To make monopolar spindles, 5 µM S-trityl-l -cysteine (STLC; Sigma-Aldrich) was added 15 min before imaging (10 mM stock). To rigor CenpE to microtubules, 90 nM GSK-923295 (MedChem Express) was added 15 min before imaging (30 µM stock; Magidson et al., 2015 (link)). To visualize tubulin as a third color, 100 nM SiR-tubulin dye (Cytoskeleton, Inc.) was added 1 h before imaging (1 mM stock) along with 10 µM verapamil (10 mM stock; Cytoskeleton, Inc.) to prevent dye efflux.
Gsk 923295
Manufactured by MedChemExpress
Sourced in China
GSK-923295 is a laboratory compound developed by MedChemExpress for research purposes. It is a small molecule that functions as a selective inhibitor. The core function of GSK-923295 is to serve as a tool compound for scientific investigation. No further details on intended use or application can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Zm447439, by Bio-Techne (2 mentions)
Ro 3306, by Selleck Chemicals (2 mentions)
Mln8237, by Selleck Chemicals (2 mentions)
Abt 751, by MedKoo Biosciences (2 mentions)
Nocodazole, by MedChemExpress (2 mentions)
Verapamil, by Cytoskeleton (1 mentions)
Sir tubulin dye, by Cytoskeleton (1 mentions)
S trityl l cysteine stlc, by Merck Group (1 mentions)
Umk57, by Aobious (1 mentions)
Reversine, by Merck Group (1 mentions)
Cofilin, by Santa Cruz Biotechnology (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Bubr1, by Abcam (1 mentions)
Nsc95397, by Merck Group (1 mentions)
Blebbistatin, by Merck Group (1 mentions)
Bi2536, by Merck Group (1 mentions)
Ipa 3, by Merck Group (1 mentions)
Sb743921, by Selleck Chemicals (1 mentions)
Ccnb1, by Merck Group (1 mentions)
Itx 3, by Merck Group (1 mentions)
9 protocols using gsk 923295
1
Monopolar Spindle Formation and Visualization
2
Mitotic Spindle Inhibitors Screening
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The CENP-E inhibitor GSK-923295 (MedChemExpress LLC # HY-10299) was used at 200 nM. NOC (VWR #80058-500) was used at 100–500 ng/ml. STLC (Tocris #2191) was used at 25 μM. ProTAME (Concept Life Sciences custom synthesis) was used at 25 μM. Okadaic acid (LC Labs O-5857) was used at 200 nM. RO-3306 (Selleck #S7747) was used at 10 μM. AZ3146 (R&D Systems #3994/10) was used at 2μM. BI2536 (Synthesized in-house) was used at 100 nM. MLN8237 (Selleck #S1133) was used at 10–50 nM. ZM447439 (Tocris Bioscience #2458/10) was used at 2 μM. UMK57 (Aobious #AOB8668) was used at 100 nM.
3
Cell Treatment and Sample Preparation
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All cell treatments were done in standard 12-well plates. HCT-116 cells were seeded at 170k cells per well and K562, Mv4;11, MOLM-13 cells at a density of 250k cells per well. After 1 day, the cells were treated with compounds for the indicated period of time. The adherent cells were washed thrice with ice cold PBS after which they were scraped from the wells and centrifuged at 8.5k × g for 15 minutes at 4°C. The suspension cells were collected by centrifugation (12k × g for 5 minutes), and washed by resuspending them in cold PBS and collectinon by centrifugation (12k × g for 5 minutes) three times. The PBS was removed and the cell pellets were stored at −80°C and either used for LC/MS analysis or Western blotting.
The compounds ABT-751 (MedKoo Biosciences, Inc.), Nocodazole (MedChem Express), and GSK-923295 (MedChem Express) were purchased.
The cells for RNA extraction and qPCR were not washed but directly collected in 1 mL TRI Reagent (TR 118) and stored at −80°C.
The compounds ABT-751 (MedKoo Biosciences, Inc.), Nocodazole (MedChem Express), and GSK-923295 (MedChem Express) were purchased.
The cells for RNA extraction and qPCR were not washed but directly collected in 1 mL TRI Reagent (TR 118) and stored at −80°C.
4
High-Throughput Screening of Mitotic Regulators
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The drugs used for screening in this study are listed in Supplementary Table 1 .
Reversine, SP600125, ZM 447439, BI 2536, STLC, Dimethylenastron, SB203508, RO 3306, Cdk1 Inhibitor III, Roscovitine, NSC 95397, IPA3, Y-27632, ITX-3, Nocodazole, Paclitaxel, Blebbistatin, Cytochalasin B, MG 132 and Velcade were purchased from Sigma–Aldrich (St. Louis, MO, USA). AZ 3146 and Mps-BAY2a were obtained from Tocris (Bristol, United Kingdom). MLN8237 (Alisertib), AZD1152-HQPA (Baraserib) and SB743921 were purchased from Selleckchem. GSK923295 is from MedChem express.
Monoclonal antibodies against α-tubulin, CDK1, AURKA, PLK1, KIF11 (Sigma-Aldrich); CCNB1, CCNE1, TP53, CDKN1A, JNK1, JNK2 (Cell signaling); EB1, COFILIN (Santa Cruz), TTK, BUBR1 (Abcam). Polyclonal antibodies against PRC1 (Santa Cruz), Tpx2 [69 (link)]; Kif23 [70 (link)] were used.
Reversine, SP600125, ZM 447439, BI 2536, STLC, Dimethylenastron, SB203508, RO 3306, Cdk1 Inhibitor III, Roscovitine, NSC 95397, IPA3, Y-27632, ITX-3, Nocodazole, Paclitaxel, Blebbistatin, Cytochalasin B, MG 132 and Velcade were purchased from Sigma–Aldrich (St. Louis, MO, USA). AZ 3146 and Mps-BAY2a were obtained from Tocris (Bristol, United Kingdom). MLN8237 (Alisertib), AZD1152-HQPA (Baraserib) and SB743921 were purchased from Selleckchem. GSK923295 is from MedChem express.
Monoclonal antibodies against α-tubulin, CDK1, AURKA, PLK1, KIF11 (Sigma-Aldrich); CCNB1, CCNE1, TP53, CDKN1A, JNK1, JNK2 (Cell signaling); EB1, COFILIN (Santa Cruz), TTK, BUBR1 (Abcam). Polyclonal antibodies against PRC1 (Santa Cruz), Tpx2 [69 (link)]; Kif23 [70 (link)] were used.
5
Preparation of Inhibitor Stock Solutions
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Inhibitors of CENPE (GSK923295), BCL-2/BCL-XL (navitoclax), and MPS-1 (BAY1217389) were obtained from MedChem Express (Shanghai, China) and were reconstituted in sterile dimethyl sulfoxide (DMSO, Sigma-Aldrich Co., Ltd., St. Louis, MO, USA) to a stock concentration of 5 or 10 mM. Several aliquots were prepared and stored at −20 °C to avoid repeated cycles of freezing and thawing and, consequently, the loss of compounds’ activity. For each independent experiment, a work solution was prepared in fresh culture medium to prepare the desired concentrations.
6
Xenopus and HeLaM Cell Experiments
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For Xenopus experiments, STLC (Sigma-Aldrich; 50 mM stock in DMSO) was diluted to 1 mM in PBS and injected into the blastocoel of MO-treated embryos 21.5 h after fertilization (16°C). The needle volume was set to 18 nl, and each embryo was injected twice, into each side of the blastocoel. Control embryos were injected with the same volumes of DMSO diluted 1:50 in PBS. Embryos were incubated at RT for 2 h before fixation.
For HeLaM drug treatments, cells were transfected with scrambled or LIC1 and 2 siRNAs and incubated for 70–72 h. Drug treatments were performed in DMEM + 10% FCS at 37°C followed by fixation and immunofluorescence analysis. Eg5 was inhibited with 2 µM STLC for 2 h. CENP-E was inhibited with 100 nM GSK-923295 (10 mM stock in DMSO; Medchem Express; Wood et al., 2010 (link)) for 90 min.
For HeLaM drug treatments, cells were transfected with scrambled or LIC1 and 2 siRNAs and incubated for 70–72 h. Drug treatments were performed in DMEM + 10% FCS at 37°C followed by fixation and immunofluorescence analysis. Eg5 was inhibited with 2 µM STLC for 2 h. CENP-E was inhibited with 100 nM GSK-923295 (10 mM stock in DMSO; Medchem Express; Wood et al., 2010 (link)) for 90 min.
7
Culturing Engineered Cell Lines for Centromere Analysis
8
Inhibitor Cocktail for Mitotic Spindle Dynamics
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The CENP-E inhibitor GSK-923295 (# HY-10299; MedChemExpress LLC) was used at 200 nM. NOC (#80058-500; VWR) was used at 100–500 ng/ml. STLC (#2191; Tocris) was used at 25 μM. ProTAME (Concept Life Sciences custom synthesis) was used at 25 μM. Okadaic acid (O-5857; LC Labs) was used at 200 nM. Doxycycline hyclate (#24390-14-5; Sigma-Aldrich) was used at 2 μg/ml. RO-3306 (#S7747; Selleck) was used at 10 μΜ. ΑΖ3146 (#3994/10; R&D Systems) was used at 2 μM. ΒΙ2536 (synthesized in-house) was used at 100 nM. MLN8237 (#S1133; Selleck) was used at 50 nM. ZM447439 (#2458/10; Tocris Bioscience) was used at 2 μM. Calyculin A (#9902S; Cell Signaling Technology) was used at 5 nm. Palbociclib (#PD-0332991; Selleck) was used at 500 nM.
9
Cell Treatment and Sample Preparation for LC-MS and Western Blot Analysis
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All cell treatments were done in standard 12 well plates. HCT-116 cells were seeded at 170k cells per well and K562, Mv4;11, MOLM-13 cells at a density of 250k cells per well. After 1 d, the cells were treated with compounds for the indicated period of time. The adherent cells were washed thrice with ice cold PBS after which they were scraped from the wells and centrifuged at 8.5k g for 15 min at 4 °C. The suspension cells were collected by centrifugation (12k g for 5 min), and washed by resuspending them in cold PBS and collectinon by centrifugation (12k g for 5 min) three times. The PBS was removed and the cell pellets were stored at -80 °C and either used for LC-MS analysis or Western blotting. The compounds ABT-751 (MedKoo Biosciences, Inc.), Nocodazole (MedChem Express) and GSK-923295 (MedChem Express) were purchased. The cells for RNA extraction and qPCR were not washed but directly collected in 1 mL TRI Reagent ® (TR 118) and stored at -80 °C.
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