Embryos from mice or rats were harvested on day 11.5 or 13 of gestation, respectively (vaginal plug positive=day 0). Lungs were dissected and cultured as described (Jesudason et al., 2000 (link)). Cyclopiazonic acid (CPA) (Sigma-Aldrich Company Ltd., Dorset, UK or equivalent) was filter sterilized and added for final concentrations of 2–20 µM. Lung morphometry was assessed with terminal bud count. Peristaltic wave frequency was measured in 10 min periods (Jesudason et al., 2005 (link)). At the end, lung cultures were homogenized for RNA extraction or prepared for histology. Mitotic cells were labeled with Anti-phospho-Histone H3 Ser10 staining (Brand and Perrimon, 1993 (link)). Epithelial tip cultures were performed as described, but without enzymatic digestion (Bellusci et al., 1997 (link)). Mechanotransduction inhibitors used included: PKCi (Bisindolylmaleimide I Hydrochloride, #203290 Calbiochem), PLCi (L108 Edelfosin, #BML-L108, Enzo Life Science), ROCKi (Y26732, #Y0503, Sigma), RACi (NSC 23766, #553502, Millipore).
Nsc23766
Manufactured by Merck Group
Sourced in United States, Germany, Morocco
NSC23766 is a small molecule laboratory reagent used in research applications. It functions as an inhibitor of the Rac guanine nucleotide exchange factor. The core function of this product is to modulate cellular signaling pathways involving the Rac protein.
Automatically generated - may contain errors
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79 protocols using nsc23766
1
Lung Morphogenesis Assay in Vitro
2
Generating Stable Knockdown Cell Lines
3
Inhibiting Cytoskeletal Regulators in Cell Culture
4
Endothelial Barrier Regulation Assay
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Unless otherwise stated, all reagents were purchased from Sigma-Aldrich (Dublin, IRL). Cytokines (TNF-α, IL-6), apocynin and NSC23766 were purchased from Millipore (Cork, IRL). Primary antisera were purchased from the following sources: Anti-occludin IgG, anti-claudin-5 IgG, and anti-ZO-1 IgG (Bio-Sciences, Dublin, IRL); Anti-VE-cadherin IgG (Abcam, Cambridge, UK); Anti-gp91 IgG, anti-p47 IgG, and anti-GAPDH IgG (Santa Cruz Biotechnology, CA, USA); HRP-conjugated secondary antisera for VE-cadherin, occludin, claudin-5, and GAPDH were purchased from Cell Signalling Technologies Inc. (MA, USA). HRP-conjugated secondary antisera for gp91 and p47 were purchased from Sigma Aldrich. siRNA constructs for gp91 (SC35503, RefSeq NM_000397.3) and p47 (SC29422, RefSeq NM_000265.5) were obtained from Santa Cruz Biotechnology.
5
Rho GTPase Signaling Inhibition
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Rac1 inhibitor NSC23766 was used at 50 μM according to manufacturer's instructions and IC50 reported in the literature (Millipore) (Gao et al., 2004 ). ROCK inhibitor Y‐27632 was used at 10 μM according to manufacturer's instructions (Millipore). For adenoviral studies, constitutively active and dominant negative RhoA and Rac1 adenovirus were purchased, prepared, and titrated according to manufacturer's instructions (CellBioLabs).
6
Electroporation-Mediated Gene Delivery in Gerbil Molar Development
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NSC23766 (Millipore) was dissolved in H2O and diluted in DMEM with Glutamax (Gibco) and 10% FBS to obtain a final concentration of 90 μM. A total of 300 nM RhoA-siRNA (Santa Cruz Biotechnology) was delivered by Lopofectamine® 2000 (Life Technologies) reagent according to the manufacturer’s instructions.
For electroporation, Fast Green (Sigma, 1:1000) was added to both of the plasmid solutions of pcDNA-EGFP-RhoA-Q63L and pcDNA-EGFP for visualization within the tissue. A microcapillary needle was used to inject 1 μg/μL DNA into the dental epithelium of gerbil molars at E22.0, followed by four pulses at 40 V applied using an electroporator for 50 milliseconds with 1 second intervals.
Mandibular molar regions were obtained from E22.0 gerbil and E13.0 mouse embryos. Molar explants were cultured in vitro using the Trowell technique. Ten specimens were examined via the morphological and IF analyses.
For electroporation, Fast Green (Sigma, 1:1000) was added to both of the plasmid solutions of pcDNA-EGFP-RhoA-Q63L and pcDNA-EGFP for visualization within the tissue. A microcapillary needle was used to inject 1 μg/μL DNA into the dental epithelium of gerbil molars at E22.0, followed by four pulses at 40 V applied using an electroporator for 50 milliseconds with 1 second intervals.
Mandibular molar regions were obtained from E22.0 gerbil and E13.0 mouse embryos. Molar explants were cultured in vitro using the Trowell technique. Ten specimens were examined via the morphological and IF analyses.
7
Generating Stable Knockdown Cell Lines
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All mouse HCC cell lines were cultured in high-glucose DMEM (Life
Technologies) with 10% FBS and 1% Pen/Strep. All mammalian cell lines were grown
at 37°C. Human HCC cell lines were obtained from ATCC. All other cell
lines were from Lewis lab collections.
DF1 chicken fibroblast cells were cultured in growth medium at
39°C in high-glucose DMEM (Life Technologies) with 10% FBS and 1%
Pen/Strep.
Stable knockdown cell lines were generated from lentiviral delivery of
shRNAs directed to a mouse target mRNA. shRNA IDs are detailed inSupplemental Table 3 .
The RAC1 inhibitor NSC23766 (Millipore, 553502) was added to cells as
they were plated in a transwell migration chamber to a final concentration of
50μM.
Technologies) with 10% FBS and 1% Pen/Strep. All mammalian cell lines were grown
at 37°C. Human HCC cell lines were obtained from ATCC. All other cell
lines were from Lewis lab collections.
DF1 chicken fibroblast cells were cultured in growth medium at
39°C in high-glucose DMEM (Life Technologies) with 10% FBS and 1%
Pen/Strep.
Stable knockdown cell lines were generated from lentiviral delivery of
shRNAs directed to a mouse target mRNA. shRNA IDs are detailed in
The RAC1 inhibitor NSC23766 (Millipore, 553502) was added to cells as
they were plated in a transwell migration chamber to a final concentration of
50μM.
8
Modulating MSC Migration via Pathway Inhibitors
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In order to influence the migration of MSCs, AMD3100 (CXCR4 antagonist), Y-27632 (inhibitor of Rho-ROCK pathway), and NSC23766 (inhibitor of Rac pathway) were applied throughout all channels. Each of the inhibitors Y-27632 (25μM, Sigma-Aldrich, St. Louis, MO, USA), NSC23766 (50μM, Sigma Aldrich, St. Louis, MO, USA), and AMD3100 (25μg/ml, Sigma Aldrich, St. Louis, MO, USA) were mixed in 2:1 EGM-2 and DMEM with addition of SDF-1α (250 ng/ml) on the condition channel. A total of five groups including conditions with media only (no SDF-1α, no drugs), SDF-1α only group, and three inhibitor-treated groups were tested. Media for each group were replaced every 24 hours, and images were obtained with an EVOS fluorescence microscope (EVOS® FL Auto, Life Technologies, USA).
9
Inhibitor Effects on Cell Migration
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To determine the effect of inhibitors on cell migration, +/− cells were transplanted into sublethally irradiated NSG recipients. At 4 days after transplantation, cells were imaged by intravital microscopy. After 2 to 3 hr of imaging, NSC 23766 (3 mg/kg, Sigma), Bio 5192 (1 mg/kg, Tocris), or AMD3100 (1 mg/kg, Sigma) were injected IV into the mouse, and imaging was resumed 30 min later for up to 4-hr post-injection.
10
In vitro Cell Migration Assay
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Cell migration was measured by in vitro scratch assay. 5 × 105 cells were plated in a 12-well plate one day before transfection. When cells reached 95~100% of confluency in MEM with 10% FBS, the medium was replaced with fresh serum-free MEM to inhibit further proliferation. At least 24 hours after transfection (72 hours after transfection for knockdown conditions), the cell monolayer was scraped in a straight line to create a “scratch” with a p1000 pipet tip. The number of migrated cells was counted at 10 hours after scratch using image J software. When applicable, SH-SY5Y cells are treated with actinomycin-D (1 μM, Sigma Aldrich) for 24 hours ahead of scratch or NSC23766 (Rac1 inhibitor, 50 μM, Sigma Aldrich) for 12 hours ahead of scratch.
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