U87-NT cells were seeded at 3 × 103/well in low adherence 96 well plates (ThermoFisher, Altrincham, UK) as previously described [14 (link)]. Three days after seeding, spheroids contained within the wells were embedded in rat tail collagen V (Corning Life Science, Glendale, AZ 85301, USA), polymerisation was achieved with 1 M NaOH. The inhibitors CCG-1423 and Rhosin-HCl (Tocris, Bristol, UK) were resuspended in DMSO and added at a predetermined anti-migratory concentration (CCG-1423: 500 nM, Rhosin: 1 µM). Control spheroids were mock-treated with DMSO-supplemented medium only. Spheroids were allowed to grow for 72 h in collagen, fixed with 4% PFA and labelled with Phalloidin-TRITC (ThermoFisher, Altrincham, UK) in their collagen plugs. Image z-stacks of spheroids were acquired on a Zeiss 880 LSM confocal microscope (Zeiss, Birmingham, UK) with an EC plan-neofluar 10× objective. For analysis maximum projections of the z-stacks were used. The resulting grey-scale images were inverted and using the free-hand selection tool in ImageJ, the outline of the spheroid as well as of the gaps between migration cells were drawn and the area measured (for illustration see Supplement Figure S2 ).
Ccg 1423
Manufactured by Bio-Techne
Sourced in United Kingdom
CCG-1423 is a lab instrument developed by Bio-Techne. It is designed for performing cell culture growth analysis.
Automatically generated - may contain errors
Lab products found in correlation
Rhosin hcl, by Bio-Techne (1 mentions)
Rat tail collagen 5, by Corning (1 mentions)
Phalloidin tritc, by Thermo Fisher Scientific (1 mentions)
Lsm 880 confocal microscope, by Zeiss (1 mentions)
Rhosin, by Bio-Techne (1 mentions)
6 well tc plates, by Corning (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Corning (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Cacl2, by Thermo Fisher Scientific (1 mentions)
Plasmocin, by Thermo Fisher Scientific (1 mentions)
6 mm punch, by Integra LifeSciences (1 mentions)
Zcl278, by Bio-Techne (1 mentions)
Eht1864, by Bio-Techne (1 mentions)
Tomatidine, by Selleck Chemicals (1 mentions)
Tolcapone, by Selleck Chemicals (1 mentions)
Ku 60019, by Selleck Chemicals (1 mentions)
Lat a, by Bio-Techne (1 mentions)
6 bromoindirubin 3 oxime, by Selleck Chemicals (1 mentions)
6 protocols using ccg 1423
1
Spheroid Migration Assay in 3D Collagen
2
Skin Explant Culture to Assess CCG-1423 Effects
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C57BL/6J mice were sacrificed at p26, backs shaved, skin removed, and then 6-mm punch (Integra; 33-36) was used to generate circular punches, which were subsequently attached to the bottom of 6-well TC plates (Corning; 3516) with 6uL of Matrigel (Corning; 356237). Explants were cultured in 154CF media (Gibco; M15CF500) supplemented with 15% chelated FBS (GE HyClone; SH30396.03), 1% Penicillin Streptomycin (Gibco; 15140-122), 0.05 mM CaCl2 (Gibco; 50-9702), 0.4% Amphotericin B (Gibco; 15290026), 0.01% Plasmocin (Invitrogen; NC9886956), and 1% HKGS (Gibco; S-0001-5). Paired explants from same mouse were either treated with 150 μm CCG-1423 (Tocris; 5277) or DMSO control. Explants were cultured for 72 h, then fixed in 4% PFA for 24 h and imbedded in paraffin blocks for H&E and immunofluorescence staining.
3
Inhibitor Effects on Cell Spreading
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Inhibitor experiments were performed in serum-containing medium, and treatments began 5 minutes prior to plating (spreading) or 5 minutes prior to scratch-wounding. Small-molecule inhibitors against signaling proteins, CDC42 (ZCL 278, 55 μM in DMSO), ERK (FR 180204, 3 μM in DMSO), FAK (PF 573228, 40 nM in DMSO), PI3K (LY294002, 5 μM in DMSO), RAC (EHT 1864, 600 nM in DMSO), and RHOA (CCG 1423, 3 μM in DMSO) were obtained from Tocris Bioscience, Bristol, UK. In order to ensure specificity of the inhibitors, dose-range studies were conducted with 3 doses for each inhibitor. Concentrations were chosen based on the lowest concentration for significant effect on spreading on any matrix. All inhibitors were used at a concentration less than 5x of the reported In vitro ID50 for each molecule.
4
Modulation of MRTF-A/B and Actin Dynamics
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In Figure 2H , human coronary artery SMCs were transduced with MRTF-A virus or MRTF-B virus (200 MOI) in 1% Smooth Muscle Differentiation Supplement (SMDS, Life Technologies, S-008-5) M231 medium for 96h. Subsequently, the medium was exchanged for fresh 1% SMDS medium, and 10μM CCG-1423 (Tocris Bioscience, #5233) or the corresponding volume of DMSO (Sigma-Aldrich, #D5879) added directly the medium. Cells were then harvested after an additional 72h.
To depolymerize actin in human coronary artery endothelial cells, Latrunculin B (Lat B, 100nM, Calbiochem, #428020) or DMSO (Sigma-Aldrich, #D5879) was added at 96h after transducing with MRTF-B virus, and cells were harvested after additional 24h. Before adding the LatB, cells were also transferred to low-serum medium (2.5% FBS) for a 24h period.
To inhibit the YAP-TEAD interaction, 2μM verteporfin (Sigma-Aldrich, SML0534-5MG) or the corresponding volume of DMSO (Sigma-Aldrich, #D5879) was added to the medium after transduction with MRTF-B virus for 72h (human coronary artery SMCs). Cells were harvested for RNA extraction after an additional 24h.
To depolymerize actin in human coronary artery endothelial cells, Latrunculin B (Lat B, 100nM, Calbiochem, #428020) or DMSO (Sigma-Aldrich, #D5879) was added at 96h after transducing with MRTF-B virus, and cells were harvested after additional 24h. Before adding the LatB, cells were also transferred to low-serum medium (2.5% FBS) for a 24h period.
To inhibit the YAP-TEAD interaction, 2μM verteporfin (Sigma-Aldrich, SML0534-5MG) or the corresponding volume of DMSO (Sigma-Aldrich, #D5879) was added to the medium after transduction with MRTF-B virus for 72h (human coronary artery SMCs). Cells were harvested for RNA extraction after an additional 24h.
5
Inhibitor Compounds for Research
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Tolcapone (S4021), etretinate (S4699), KU-60019 (S1570), tomatidine (S9430), and citalopram HBr (S4749) were obtained from Selleckchem. Z-VEID-FMK (ab142025) was obtained from Abcam. CCG-1423 (5233) was obtained from Tocris. SCH-23390 hydrochloride (HY-19545A) was obtained from MedChemExpress. N-Butylidenephthalide, (E) + (Z) (sc-279727) was obtained from Santa Cruz Biotechnology. Prior to use, all inhibitors were resuspended in dimethyl sulfoxide (DMSO), with the exception of n-Butylidenephthalide which was resuspended in ethanol.
6
3D Glioma Spheroid Migration Assay
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The glioma cell lines U251 and U87 were grown in vitro as previously described [11] (link). Spheroids were generated from the cells in low adherent 96-well plates (Nunc, UK) as previously described [12] (link), embedded in collagen and treated with a panel of small molecule inhibitors at predetermined anti-migratory concentrations, including 6-bromoindirubin-3-oxime (5 µM) (BIO, Selleckchem), latrunculin A (10 µM) (lat A, Tocris) and CCG-1423 (500 nM) (Tocris). Glioma cells emanating away from the original spheroid cores were allowed to migrate into collagen for 72 h. After completion of the experiment, the whole collagen plugs containing spheroids and migrating cells were fixed with PFA (4% in PBS, overnight at 4°C).
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