Cells were seeded at a density of 2.1 × 104 cells per cm2 in the appropriate basal medium for all inhibitor studies described in this section, experiments began following overnight adherence. hTERT-MSCs were treated with an ILK inhibitor, ILKi (Cpd-22; 0.6, 1, 2.5 μM; Merck), EGFR inhibitor, EGFRi (PD153035; 0.6, 1, 1.4 μM; Santa Cruz) or PDGFR inhibitor, PDGFRi (sc-222142; 0.3, 0.5, 0.8 μM; Santa Cruz) for 24 h. Primary MSCs and HDFs were incubated in DMEM supplemented with 1% P/S and 0.5% FBS (synchronisation medium) for 24 h, before a further incubation (15 min or 6 h) with recombinant EGF protein (1-100 nM, Cell Guidance Systems). For RGD studies, primary MSCs were incubated in synchronisation medium for 24 h. Samples were incubated overnight with 1 μM ILKi or vehicle control after which RGD integrin binding peptide (100 μg/ml, Anaspec) was added to samples for 6 h. For growth factor plus ILKi studies, primary MSCs were incubated overnight with 1 μM ILKi or vehicle control before a further incubation period of 24 h untreated, or treated with 1 μM ILKi, 10 nM recombinant EGF, PDGF (20 nM, Cell Guidance Systems) alone or in combination.
Cpd22
Manufactured by Merck Group
Sourced in United States, Germany
Cpd22 is a lab equipment product from Merck Group. It is designed for use in scientific research and analysis. The core function of Cpd22 is to facilitate the controlled and precise handling of small liquid volumes.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Mk 2206, by Selleck Chemicals (2 mentions)
Recombinant human wnt3a, by R&D Systems (2 mentions)
Pdgfri, by Santa Cruz Biotechnology (1 mentions)
Cariporide, by Santa Cruz Biotechnology (1 mentions)
Pf573228, by Merck Group (1 mentions)
Anti β actin antibody, by Cell Signaling Technology (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Dulbecco s modified eagle s medium dmem high glucose, by Corning (1 mentions)
Losartan potassium, by Bio-Techne (1 mentions)
Cilengitide, by Bio-Techne (1 mentions)
Ym155, by MedChemExpress (1 mentions)
Ly294002, by Bio-Techne (1 mentions)
Sb431542, by Cayman Chemical (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Cholera toxin, by Merck Group (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
19 protocols using cpd22
1
Inhibitor Effects on MSC Differentiation
2
Exploring Focal Adhesion Kinase Pathways
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hASC‐HLCs were seeded on EC matrix at a density of 75,000/cm2. After a 6‐hr incubation, focal adhesion kinase (FAK) inhibitor (1 μM PF228; Selleckchem, Houston, TX, USA), Src family kinase (SFK) inhibitor (5 μM, PP2, Selleckchem) and integrin‐linked kinase (ILK) inhibitor (1 μM Cpd22, Merck Millipore, Darmstadt, Germany) were added separately for 15 hrs; then, the cells were cultured for 72 hrs, and the metabolic properties of the hASC‐HLCs were analysed further.
3
Inhibiting ILK and NHE1 in Cancer Cells
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MDA-MB-231 cells [45 (link)] and PC-3 cells [46 (link)] were cultured as previously described. The ILK inhibitor, Cpd22, was purchased from EMD Millipore (Burlington, MA, USA). The NHE1 inhibitor (cariporide) and β1-integrin activating (P4G11) and inhibitory (P5D2) antibodies were purchased from Santa Cruz Biotechnology, (Santa Cruz, CA, USA).
4
Investigating IFN-α and Integrin Signaling
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Human IFN-α was obtained from Merck KGaA. The β1-integrin function-blocking antibody was purchased from EMD Millipore (Billerica, MA, USA; cat. no. MABT821). The rabbit polyclonal anti-IFN-stimulated gene (ISG) 15 (cat. no. 2743S) and anti-protein kinase R (PKR; cat. no. 3072S) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The antibody against the HCV core protein (cat. no. ab2740) and HCV nonstructural protein (NS) 5A (cat. no. ab13833) were purchased from Abcam (Cambridge, UK). The rabbit polyclonal anti-β-actin antibody (Cell Signaling Technology, Inc.; cat. no. 4967) was used as a control. Anti-rabbit horseradish peroxidase (HRP) conjugated IgG (cat. no. 7074; Cell Signaling Technology, Inc.) was used as the secondary antibody. The integrin-linked kinase (ILK) inhibitor Cpd 22 was purchased from EMD Millipore, and the focal adhesion kinase (FAK) inhibitor PF 573228 was from Sigma-Aldrich (Merck KGaA).
5
Rat Neonatal H9c2 Cardiac Myoblast Cell Line Protocol
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The rat neonatal H9c2 cardiac myoblast cell line was obtained from the Korea Cell Line Bank (Seoul, Korea). The cells were cultured in Dulbecco’s modified Eagles medium (DMEM)/high glucose with 10% fetal bovine serum and 1% penicillin-streptomycin (Corning/Mediatech, Inc., Manassas, VA, USA,).
DOX was purchased from Tocris Bioscience (Cat#2252, Bristol, UK). LY294002 (Cat#1130), the PI3K inhibitors, cilengitide (Cat#5870), and losartan potassium (Cat#3798), the integrin inhibitors αvβ3 and αvβ5, and the angiotensin II type 1 receptor (AT1R) inhibitors respectively, were purchased from Tocris Bioscience. The ILK inhibitor Cpd22 (Cat#407331) was obtained from EMD Millipore Corp. (Darmstadt, Germany). The survivin inhibitor YM-155 was purchased from MedChem Express (Cat#HY-10194, Monmouth Junction, NJ, USA).
DOX was purchased from Tocris Bioscience (Cat#2252, Bristol, UK). LY294002 (Cat#1130), the PI3K inhibitors, cilengitide (Cat#5870), and losartan potassium (Cat#3798), the integrin inhibitors αvβ3 and αvβ5, and the angiotensin II type 1 receptor (AT1R) inhibitors respectively, were purchased from Tocris Bioscience. The ILK inhibitor Cpd22 (Cat#407331) was obtained from EMD Millipore Corp. (Darmstadt, Germany). The survivin inhibitor YM-155 was purchased from MedChem Express (Cat#HY-10194, Monmouth Junction, NJ, USA).
6
Inhibition of Signaling Pathways in Cell Culture
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Unless otherwise stated, all chemicals and plastics were obtained from Merck (Darmstadt, Germany). An inhibitor of Smad 2/3 (SB 431542) was purchased from the Cayman Chemical Company (Ann Arbor, MI, USA); an inhibitor of ILK (Cpd22) was purchased from Merck; and antibodies against EGF (cat. no. AF236), HGF (cat. no. MAB294), IGF-1 (cat. no. AF-291-NA), and TGF-β1 (cat. no. AF-101-NA) were purchased from R&D Systems (Abingdon, UK).
7
Culturing High-Grade Meningioma Cells
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The IOMM-Lee high-grade meningioma cell line was obtained from ATCC (CRL-3370). Cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. Cpd22® (Merck Millipore, Burlington, MA, USA) was dissolved in DMSO to prepare the drug solution. Cells were cultured in a CO2 incubator under standard conditions of 5% CO2, 95% humidity, and 37 °C temperature. Once the cells reached the desired confluency (70–80%), the cells were cleared by trypsinization and transferred to new culture vessels for maintenance.
8
Meningioma Primary Cell Inhibition Assay
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The primary cells were procured from the Tissue Bank, Imperial College London. The primary cells were seeded from surgically resected tissues of meningioma patients. The cells were passaged prior to freezing in liquid Nitrogen for long term storage. The cells were taken out and revived in DMEM-F12 media. However, owing to the slow growth (doubling time was nearly 50 h) the cells were moved to Waymouth media supplemented with 20% FBS. For the inhibition assay cells were passaged in 6 well plates in triplicates and treated with 2.5 μM of ILK inhibitor namely Cpd22, Merck Millipore. The cells were seeded in two batches “Untreated” (No drug) and “Treated” (With Drug). The treatment was carried out for 24 h. Post-treatment the cells were pelleted down and further taken forward for RNA extraction.
9
Ischemia-Reperfusion Injury in Isolated Hearts
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All hearts were subjected to a 20-min stabilization period, 30-min regional ischemia, and 30-min reperfusion. The hearts were randomly divided into individual groups, a sham group, I/R group, and I/R with reagent treatment group. In the sham group, the suture was not tightened and hearts were perfused constantly with KHS for 80 min without any treatment. In the I/R group, the hearts were stabilized for 20 min, then the LAD was occluded for 30 min to induce regional ischemia, followed by 30-min reperfusion with KHS. The reagents included ILK agonist L-α-phosphatidyl-D-myo-inositol 3,4,5-triphosphate,dioctanoyl (LPTP) (10−7 M) (Sigma, St. Louis, MO), ILK inhibitor Cpd22 (6 × 10−7 M) (Millipore, St Charles, MO), and the Akt inhibitor MK2206 (7 × 10−8 M) (Selleckchem, Houston, TX). Reagents were dissolved in KHS and infused before inducing ischemia. When an inhibitor and agonist were infused into the same isolated heart, the inhibitor was introduced 10 min before the agonist.
10
Mammary Epithelial Cell Culture Protocols
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NMuMG mouse mammary epithelial cells (ATCC) were cultured in high glucose DMEM (Hyclone) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin (Life Technologies), and 100 μg/ml streptomycin (Life Technologies). SCp2 mouse mammary epithelial cells were cultured in DMEM:F12 supplemented with 2% FBS, 10 µg/ml insulin (Sigma), and 50 µg/ml gentamicin (Life Technologies); 4T1 mouse mammary carcinoma cells (ATCC) were maintained in RPMI medium (Hyclone) supplemented with 10% FBS and 50 µg/ml gentamicin. MCF10A human mammary epithelial cells were cultured in DMEM:F12 supplemented with 5% horse serum (Fisher Scientific), 100 ng/ml cholera toxin (Sigma), 10 µg/ml insulin, 20 ng/ml epidermal growth factor (Sigma), 0.5 mg/ml hydrocortisone (Fisher Scientific), and 50 µg/ml gentamicin. Cells were treated for 24 h with vehicle (0.1% bovine serum albumin) or TGFβ1 (5 ng/ml; R&D Systems) starting 24 h after initial plating. For experiments using the ILK inhibitor CPD-22 (1 μM; Millipore), cells were pretreated with the inhibitor for 24 h before exposure to TGFβ1.
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