The viability of NHMs, B16F10, A375, A2058, and MC38 cells on 35 mm culture plates was measured 24 h after CAP treatment for the indicated periods for crystal violet assays according to the standard protocols recommended by the manufacturer (Sigma-Aldrich, St. Louis, MO, USA). Briefly, cells were stained with 0.1% crystal violet in 10% ethanol for 5 min at room temperature and rinsed four times with distilled water. The crystal violet retained by adherent cells was extracted with 95% ethanol, and the absorbance at 590 nm was determined using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Relative cell viability was calculated by dividing the absorbance of the experimental group by the absorbance of the control group. The anticancer effects of combinations of an EGFR inhibitor and CAP were determined by pretreating cells with the EGFR inhibitor AG1478 (Sigma-Aldrich) for 1 h, followed by treatment with CAP for 120 sec and assessment of cell viability after 24 h. After the CAP device was placed as close as possible to the culture medium, the cells were treated with CAP directly (Figure 5 ). The effects of combinations of the EGFR inhibitor AG1478 with H2O2 (Sigma-Aldrich) and SNP (Sigma-Aldrich) on cancer cell viability were assessed by pretreating cells with AG1478 for 1 h followed by treatment with H2O2 or SNP, and assessment of cell viability after 24 h.
Ag1478
Manufactured by Merck Group
Sourced in United States, Germany, Japan, Sao Tome and Principe, Macao
AG1478 is a tyrosine kinase inhibitor. It functions by inhibiting the epidermal growth factor receptor (EGFR).
Automatically generated - may contain errors
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108 protocols using ag1478
1
Evaluating Anti-Cancer Effects of Plasma and Oxidants
2
Cardiac and Signaling Pathway Modulation in Zebrafish
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To decrease cardiac contractility after MTZ (ablated) or DMSO (control) treatments, fish were immediately incubated in egg water with 1 mg/ml of tricaine (Sigma, St. Louis, MO) or 10 μM of blebbistatin (Sigma, St. Louis, MO) for 12 hr and then washed three times for analyses. To inhibit BMP or Erbb2 signaling after MTZ (ablated) or DMSO (control) treatments, fish were immediately treated with 10 μM Dorsomorphin (DM) (Sigma, St. Louis, MO) or 5 μM AG1478 (Sigma, St. Louis, MO), respectively. Fish were treated for 24, 48, or 72 hr as indicated. To inhibit BMP, Erbb2 or Notch signaling after MTZ (ablated) or DMSO (control) treatments, fish were immediately treated with 10 μM Dorsomorphin (DM) (Sigma, St. Louis, MO), 5 μM AG1478 (Sigma, St. Louis, MO), or 10 mM DAPT (Sigma, St. Louis, MO) until indicated time for each experiment.
3
AG1478 Yeast Supplement for Gene Expression
4
Ventricular Cardiomyocyte Ablation in Zebrafish
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To perform ventricular CM ablation, Tg(vmhc:mCherry-NTR) larvae were treated with 6 mM MTZ (metronidazole, Sigma-Aldrich) in E3 water for 4 h at 3 dpf as previously described [11 (link)]. To modulate signaling pathways, larvae were incubated with the following chemicals for the indicated time period: 100 μM DAPT (Sigma-Aldrich), 12 μM AG1478 (Sigma-Aldrich), 5 μM cardiomogen-1 (Sigma-Aldrich), 7.5 μM dorsomorphin (Sigma-Aldrich), 5 μM LDN193189 (Selleck), or 10 μM rapamycin (Cell Signaling Technology). To stop blood flow, larvae were treated with 1.8 mM tricaine (3-aminobenzoic acid ethyl ester, Sigma-Aldrich) or 10 mM BDM (2,3-butanedione monoxime, Sigma-Aldrich) in E3 water for the indicated time period, and then washed with fresh E3 water.
5
Investigating HB-EGF Signaling Pathways
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Recombinant human HB-EGF (PeproTech, Rocky Hill, NJ, USA) was dissolved in sterile distilled water. AG1478 (Sigma, St. Louis, MO, USA) were dissolved in 0.1% DMSO. Plumbagin (Sigma, St. Louis, MO, USA) was dissolved in DMSO to 0.1 M and stored at −20°C. Subsequent dilutions were made in cell culture medium. After 24 h culture in six-well plates at a density of 1.5 × 105 cells per well, HSC-T6 cells were separated into seven groups: (A) medium control: 24 h incubation with 10% FBS in DMEM; (B) DMSO control: same as medium control except addition of 0.1% DMSO; (C) HB-EGF (80 ng/mL, dissolved in DMEM): cells were incubated with HB-EGF for 24 h, (D) HB-EGF + AG1478 (5 μM), (E) HB-EGF + PL (2 μM), (F) HB-EGF + PL (4 μM), and (G) HB-EGF + PL (6 μM). The total RNA and cellular proteins were extracted for further analysis.
6
Inhibition of EGFR Signaling Pathways
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Palmitate (PA), PP2, TAK-242 and AG1478 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Novel EGFR inhibitor 542 was synthesized and characterized by our group. 542 and AG1478 were dissolved in DMSO for in vitro experiments and in sodium carboxyl methyl cellulose (CMC-Na) (1%) for in vivo experiments. Actived-Caspase-3 kit, Enhanced chemiluminescence reagent, and One Step TUNEL Apoptosis Assay Kit were obtained from Beyotime (Nantong, China).
7
SV40 Mesangial Cell Assay for Diabetic Nephropathy
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AG1478 and NAC were purchased from Sigma (St.Louis,MO). AG1478 was dissolved in DMSO for in vitro experiments and in sodium carboxyl methyl cellulose (CMC-Na) (0.5%) for in vivo experiments. NAC was dissolved in DMSO for in vitro experiments. SV40 MES 13 mesangial cell line was obtained from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China) and cultured in 3:1 mixture of DMEM medium (Gibco, Eggenstein, Germany) containing 5.5 mmol/L of D-glucose and Ham's F12 medium with 14mM HEPES supplemented with 10% FBS, 100U/ml of penicillin, and 100 mg/mL of streptomycin. In the high glucose-treated group (HG), cells were incubated with a DMEM medium containing 33mmol/L of D-glucose.
8
Pharmacological Inhibitor Preparation and Application
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Pharmacological inhibitors were prepared in recommended solvents according to the manufacturer’s instructions and stored at −20°C. Inhibitor treatments were performed with the indicated concentrations either during seeding and imaging or only during imaging (as stated in the legends). Inhibitors remained in the medium from their addition for the duration of the experiment. When performing stimulation experiments, TGF-β receptor inhibitor LY2109761 and solvent control were added to CM or control medium before stimulation. Inhibitors were purchased from the following manufacturers: eeyarestatin, tunicamycin (Cayman Chemical, #10012609 and #11445), FLI-06, golgicide A, AG1478, brefeldin A (Sigma-Aldrich, #SML0975, #G0923, #T4182, and #B7651), and LY2109761 (Cayman Chemical, #15409).
9
HA-VSMCs Response to LPS Signaling
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Human aortic-vascular smooth muscle cells (HA-VSMCs) (ATCC® CRL-1999) were purchased from In Vitro Technologies Life Sciences (VIC, Aus). LPS (rough strains from Salmonella enterica serotype minnesota Re 595 (Cat #L9746)), SB431542, AG1478, SB202190, UO126 and SP600125 were purchased from Sigma-Aldrich (MO, USA). GM6001, pyridoxatin and Ab1421180 were purchased from Abcam (VIC, Australia). Human recombinant transforming growth factorβ1(TGFβ1), antiphosphoSmad2 (Ser465/467), antirabbit imunoglobulinG (IgG) horseradish peroxidase (HRP) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit monoclonal IgG antibody (HRP conjugate) were purchased from Australian Bioresearch (WA, AU). Primers to 18S and PAI-1were purchased from Qiagen (VIC, AUS).
10
EGFR Kinase Inhibitors Modulate C. albicans Infection
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The EGFR kinase domain inhibitors were purchased from Santa Cruz (Gefitinib), Selleckchem (PD153035), Tocris Bioscience (AG1478) and Sigma (GW2974). Marimastat, GI-253023X, glibenclamide and Bapta-AM were purchased from Tocris Bioscience. Inhibitors were reconstituted in DMSO and aliquoted for appropriate freezer temperature storage. TR146 cells were incubated with inhibitors for 1 h prior to C. albicans infection, candidalysin exposure or mock treatment. Phospho-EGFR Tyr1068 (#3777), phospho-EGFR Tyr845 (#6963S), c-Fos (#2250S) and phospho-MKP1 (#2857S) antibodies were purchased from Cell Signalling Technology. Mouse anti-human α-actin antibody was purchased from Millipore (UK) (#MAB1501), goat anti-mouse (#115-035-062) and anti-rabbit (#111-035-144) horseradish peroxidase (HRP)-conjugated antibodies were purchased from Jackson Immunologicals (Stratech Scientific, UK). Fluorescent ErbB receptor affibodies were a kind gift from the Science and Technologies Facilities Council for use with confocal imaging work. APC anti-human EGFR antibody was purchased from BioLegend (# 352905) for use with Imagestream analyses. Biologically active EREG and EPG were purchased from Peprotech and used at 10, 25 and 50 ng/mL either individually or together at the same concentration.
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