NSC23766 (50 μM), blebbistatin (50 μM), and wortmannin (50 nM) (Tocris Bioscience), were reconstituted and stored in DMSO. Drugs were added in cell media 6 hours after initial cell seeding and left for 24 hours during imaging.
Wortmannin
Manufactured by Bio-Techne
Sourced in United Kingdom, United States
Wortmannin is a small-molecule inhibitor that targets the phosphoinositide 3-kinase (PI3K) enzyme family. It acts by irreversibly binding to the catalytic subunit of PI3K, thereby inhibiting its enzymatic activity. Wortmannin is commonly used as a research tool to investigate the role of PI3K in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Ly294002, by Bio-Techne (5 mentions)
U0126, by Bio-Techne (5 mentions)
U73122, by Bio-Techne (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Nsc23766, by Bio-Techne (2 mentions)
Bapta, by Bio-Techne (2 mentions)
Parthenolide, by Santa Cruz Biotechnology (2 mentions)
Pd98059, by Cell Signaling Technology (2 mentions)
Bapta am, by Bio-Techne (2 mentions)
Wortmannin, by Merck Group (2 mentions)
Ly294002, by Merck Group (2 mentions)
Limulus amebocyte lysate assay, by Cambrex (2 mentions)
Percoll, by Cytiva (2 mentions)
L glutamine, by Euroclone (2 mentions)
Rpmi 1640, by Euroclone (2 mentions)
Sb202190, by Bio-Techne (2 mentions)
Rapamycin, by Bio-Techne (2 mentions)
Afatinib, by Bio-Techne (2 mentions)
Gefitinib, by Bio-Techne (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
36 protocols using wortmannin
1
Inhibition of Cell Contractility
2
Hippocampal Neuron Cholesterol Modulation
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The following compounds were added to cell medium of hippocampal neurons and Hek‐293T: OSI‐906 (Selleckchem, Houston, TX ref.: S1091; 1 µM); Cholesterol oxidase (Choox; Calbiochem, San Diego, CA ref.: 228250; 10 IU/ml); human peptide purified IGF‐1 (Stem Cell Technologies, Canada #78022; 4 µM). For electrophysiology experiments we used: Insulin (Sigma‐Aldrich, St Louis, MO ref.: I0516; 0.5 µM); Quercetin (Sigma‐Aldrich, St Louis, MO ref.: Q4951; 20 µM); Wortmannin (Tocris, UK ref.: 1232; 0.5 µM); and Voriconazole (Hangzhou Dayangchem Co., China; 10 nM). Experiments for Cholesterol addition conducted in hippocampal slices were performed at 25ºC. Methyl‐β‐cyclodextrin‐Cholesterol (MβCD‐Ch) solution was prepared freshly at use concentration in ACSF, containing 30 µM Cholesterol Water‐soluble (Sigma‐Aldrich, St Louis, MO ref.: C4951) and 5 µM Cholesterol (Sigma‐Aldrich, St Louis, MO ref.: C3045).
3
U2OS Cell Shape Modulation
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U2OS control and SPECC1L-kd cells were cultured in standard DMEM high glucose media supplemented with 10% fetal bovine serum (Life technologies, Carlsbad, CA). For AJ change, 2 × 105 cells were plated on 0.1% porcine gelatin treated glass (Sigma-Aldrich, St. Louis, MO), and observed for cell shape change. Cells were collected at various indicated timepoints: 4 hours post plating (t = 1), 24 hours post plating (t = 2), confluency without cell shape change (t = 3), cell shape change (t = 4), 24 hours following cell shape change (t = 5), and 48 hours following cell shape change (t = 6) (Figs. 1 , 2 , 3 ). To modulate the PI3K-AKT pathway, cells were cultured in described concentrations of PI3K-AKT inhibitor, Wortmannin (TOCRIS Biosciences, Minneapolis, MN), or activator, SC-79 (TOCRIS Biosciences, Minneapolis, MN). Media with chemicals was replaced daily.
4
Inhibition of GPVI Signaling Pathways
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Each inhibitor was added 20 min before stimulation with LL-37 or CRAMP. BAPTA (inhibitor of intracellular calcium release); U-73122 (phospholipase C inhibitor); GF109203X (Protein kinase C inhibitor); SB203580 (p38-MAPK inhibitor), MG101 (calpain inhibitor); wortmannin (phosphoinositide 3-kinase inhibitor); Boc-MLF (FPR1-receptor inhibitor); WRW4 (FPR2-receptor inhibitor); and A438079 (P2X7 receptor inhibitor) were all obtained from Tocris (Bristol, UK). Pertussis toxin and cholera toxin (inhibitors of G-protein signaling) and Tirofiban (GPIIb/IIIa inhibitor) were from Sigma. Abciximab was from Elly Lilly (USA). Dasatinib (inhibitor of Src-family kinases), R406 (Syk Inhibitor), and Stattic (STAT3-Inhibitor) were from Selleckchem (Houston, USA). Anti-mouse GPVI antibody was from Emfret (clone JAQ1, Cat No. M011-0). The inhibitory monoclonal antibody HGP5C4 directed against human GPVI and the respective isotype control antibody RmC7H8 were generated by immunization of Lou/C rats with an adenovirally expressed human GPVI-Fc fusion protein. The latter represents a soluble form of GPVI with the extracellular domain of human GPVI fused to the human Fc domain. 4C9 and 5C4 monoclonal antibodies (immunoglobulin G1 subtype) specifically bound to GPVI-Fc but not control Fc65 (link). RmC7H8, raised in rats against an irrelevant human antigen, served as control monoclonal antibody (mAb).
5
Parthenolide Suppresses TWEAK-Induced RANTES
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PAM212 cells (5×104) were washed with PBS and pre-treated for two hours with 10 μM parthenolide (Santa Cruz), wortmannin (Tocris Biosciences, Minneapolis, MN), or PD98059 (Cell Signaling, Beverly, MA), followed by Fc-TWEAK or control Ig for 48 hours. Supernatants were analyzed by RANTES ELISA.
6
Investigating Downstream Kinase Signaling
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To investigate downstream signaling events, the following kinase inhibitors were employed: Wortmannin (upstream inhibitor of Akt), dorsomorphin dihydrochloride aka “compound C” (AMPK inhibitor), and BAPTA‐AM (Ca+2 chelator). Wortmannin, compound C, and BAPTA‐AM were purchased from Tocris Bioscience (Bristol, UK). Western blot for AKT, pAKT, and actin was performed as described above. Commercially available primary antibodies for AKT, pAKT, and secondary antibodies were used in westerns (Santa Cruz Biotechnologies, Santa Cruz, CA).
7
Pharmacological Manipulation of Neuronal Function
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Leptin (100 nM; provided by A. F. Parlow through the National Hormone and Peptide Program), Humulin (50 nM; Eli Lilly and Company), tolbutamide (200 μM; Sigma-Aldrich), tetrodotoxin (TTX; 2 μM; Tocris Bioscience), LY294002 (10 µM; Calbiochem), wortmannin (100 nM; Tocris Bioscience), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 μM; Sigma-Aldrich), AP5 (50 μM; Sigma-Aldrich), and picrotoxin (50 μM; Sigma-Aldrich) were added to the ACSF for specific experiments. All solutions were made according to the manufacturer’s specifications. Stock solutions of LY294002, wortmannin, CNQX, and picrotoxin were made by dissolution in dimethyl sulfoxide (Sigma-Aldrich). The concentration of dimethyl sulfoxide in the external solution was <0.1%. Stock solutions of leptin were made by dissolution in Dulbecco’s PBS (Gibco). Stock solutions of TTX and AP5 were made by dissolution in deionized water.
8
Pharmacological Modulators of Signaling Pathways
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Acetylcholine, norepinephrine, tetraethylammonium (TEA), 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), atropine, 3-isobutyl-1-methylxanthine (IBMX), indomethacin, pyrilamine, verapamil, and Nω-nitro-l -arginine methyl ester (L-NAME) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). LY294002 and Wortmannin was obtained from Tocris (Abingdon, UK) and Alexis Biochemicals (Lausen, Switzerland), respectively.
9
Standardized Onion Polyphenol Extraction and Characterization
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The brown outer layers of the onions were extracted three times with ethanol under reflux for 3 h. The combined filtrate was concentrated using a rotary evaporator, freeze dried, and stored at −20℃ until further analysis. The HPLC profile of the onion extract and the compounds identified from it was standardized using a validated HPLC assay [21 (link)]. Stock solutions of OPE were made by dissolving them in the appropriate amount in DMSO to a final concentration of 100 mg/ml, which were then stored at −20℃. GF109203X (GFX), H-89 dihydrochloride, and wortmannin were purchased from Tocris bioscience (Bristol, UK). Quercetin, luteolin and morin were purchased from Cayman Chemical (Ann Arbor, MI, USA). Stock solutions of chemicals were made by dissolving the appropriate powder mass of each chemical in either DMSO or distilled water, which were then stored at −80℃ until further use. Working solutions of chemicals were freshly made each day prior to experiments by dissolving the stock chemical solutions in electrophysiological saline solution.
10
Caspase-Mediated Apoptosis Pathway Modulation
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Chloroquine (CQ), N-acetyl-L-cysteine (NAC) and acridine orange hemi (Zinc chloride) salt were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Caspase-8,-9, or -3 colorimetric assay kit and Z-VAD-FMK (a pan-caspase inhibitor) were obtained from R&D systems (MA, USA). Cycletest Plus DNA kit and FITC-Annexin V were purchased from BD bioscience Pharmingen (San Jose, CA, USA). Wortmannin and U0126 were purchased TOCRIS (Bristol, UK). 2’7’-dichlorofluorescein diacetate, BAPTA-AM, Ru360 and JC-1 were obtained from Calbiochem (San Diego, CA, USA). Dihydroethidium, lipofectamine 2000, anti-Alexa Fluor 488, Fluo4-AM and BAPTA were purchased from Invitrogen (Carlsbad, CA, USA). Rhod2 and Ruthenium Red were obtained from Abcam (Cambridge, UK). The antibodies used in this study are as follows; anti-caspase-3, anti-caspase-9, anti-caspase-8, anti-PARP, anti-ATG5, anti-total ERK, anti-p-ERK (Cell signaling Technology, MA, USA), LC3B/MAP1LC3B (NOVUS Biologicals, USA), anti-p62 lck ligand (BD bioscience Pharmingen, CA, USA), HRP-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG (Santa Cruz Biotechnologies, CA, USA).
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