A769662

Manufactured by Bio-Techne

Sourced in United Kingdom, United States

A769662 is a chemical compound that functions as an activator of the AMP-activated protein kinase (AMPK) pathway. It is commonly used in cell-based and biochemical research applications to study the role of AMPK in various cellular processes.

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Lab products found in correlation

Metformin, by Merck Group (4 mentions) Compound c, by Bio-Techne (3 mentions) Aicar, by Cayman Chemical (3 mentions) Antimycin a, by Merck Group (3 mentions) Rotenone, by Merck Group (3 mentions) Compound c, by Merck Group (3 mentions) Phenformin, by Merck Group (3 mentions) 2 deoxyglucose, by Merck Group (2 mentions) Aicar, by Bio-Techne (2 mentions) Cyclohexamide, by Bio-Techne (2 mentions) Perm buffer 3, by BD (2 mentions) Phorbol myristate acetate (pma), by Merck Group (2 mentions) Lsrfortessa, by BD (2 mentions) Stain buffer, by BD (2 mentions) Cytofix buffer, by BD (2 mentions) Pe conjugated anti rabbit igg, by BD (2 mentions) Insulin, by Merck Group (2 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) L name, by Merck Group (1 mentions) Sbi 0206965, by Merck Group (1 mentions)

28 protocols using a769662

Preparation and Storage of Experimental Compounds

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AICAR (Toronto Chemical Research) was prepared at a concentration of 1.3 × 10⁻¹ M in distilled water and kept at − 20 °C until use. 2-deoxyglucose (Sigma) was prepared at a concentration of 10⁻¹ M in distilled water and used immediately. SBI-0206965 (Sigma-Aldrich), A-769662 (Tocris Bioscience) and the different modulator candidates were prepared in DMSO (Sigma). NA (Sigma Aldrich) was prepared in a saline-ascorbic solution (0.9% NaCl/0.01% ascorbic acid), Ach and L-NAME (Sigma Aldrich) were prepared in 0.9% NaCl solution. A stock solution (10⁻² M) was prepared for each of them and stored at − 20 °C until use (maximum 3 months).

Sanz-Gómez M., Aledavood E., Beroiz-Salaverri M., Lagartera L., Vega-Martín E., Gil-Ortega M., Cumella J., Pérez C., Luque F.J., Estarellas C., Fernández-Alfonso M.S, & Castro A. (2022). Novel indolic AMPK modulators induce vasodilatation through activation of the AMPK–eNOS–NO pathway. Scientific Reports, 12, 4225.

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Investigating Sorafenib and Cellular Signaling

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Sorafenib was purchased from Sigma‐Aldrich (St. Louis, MO, USA). The compound A‐769662 and capsaicin were purchased from Tocris Bioscience (Bristol, UK). The primary antibodies anti‐CD133, anti‐ALDH1A1, anti‐pAkt‐ser473, p‐mTOR, pAMPKα1‐thr172, pACC‐ser79, anti‐cyclin D1, anti‐PGC1α, and anti‐PPARγ and the antibodies against the corresponding total forms were obtained from Cell Signaling Technology (Danvers, MA, USA). The primary antibody anti‐β‐catenin was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). The primary antibody anti‐Hif‐1α was purchased from Novus (St. Louis, MO, USA). Anti‐alpha fetoprotein and peroxidase‐labelled secondary anti‐mouse IgG were purchased from Sigma‐Aldrich, and anti‐rabbit IgG was purchased from Calbiochem (San Diego, USA).

Bort A., Sánchez B.G., Mateos‐Gómez P.A., Vara‐Ciruelos D., Rodríguez‐Henche N, & Díaz‐Laviada I. (2019). Targeting AMP‐activated kinase impacts hepatocellular cancer stem cells induced by long‐term treatment with sorafenib. Molecular Oncology, 13(5), 1311-1331.

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Apoptosis Pathway Biomarkers Analysis

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Antibodies (Abs) against apoptosis inducing factor (AIF), phosphorylated AMPK (pAMPK), and total AMPK (tAMPK) were purchased from Cell Signaling (USA). Abs against endonuclease G (endo G) and α-tubulin were purchased from Abcam (UK). The anti-nitrotyrosine Ab was purchased from Upstate Biotechnology (USA). A769662 and Tocrisolve 100 were purchased from Tocris Bioscience (Bristol, UK). The 2-deoxy-D-glucose (2DG) was from Sigma-Aldrich (USA). All other reagents were purchased from Calbiochem (USA).

Hwang J.H., Kim Y.H., Noh J.R., Choi D.H., Kim K.S, & Lee C.H. (2015). Enhanced Production of Adenosine Triphosphate by Pharmacological Activation of Adenosine Monophosphate-Activated Protein Kinase Ameliorates Acetaminophen-Induced Liver Injury. Molecules and Cells, 38(10), 843-850.

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Chondrocyte culture and stimulation

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Human IL-1β was purchased from PeproTech (Rocky Hill, NJ, USA). N⁶-(1-iminoethyl)-L-lysine hydrochloride (L-NIL), forskolin, 8-Bromoadenosine-3′,5′-cyclic monophosphate sodium salt (8-Bromo-cAMP), dorsomorphin dihydrochloride, and A769662 were all purchased from Tocris Bioscience (Bristol, UK). Forskolin and A769662 were dissolved in DMSO/water (final DMSO dilution 1:200 and 1:5000, respectively), all other substances were dissolved in water. PGE₂ was from Cayman Chemical (Ann Arbor, MI, USA) and was dissolved in DMSO/water (final DMSO dilution 1:5000). Pronase E was obtained from Merck KGaA (Darmstadt, Germany), and collagenase P from Roche Diagnostics GmbH (Mannheim, Germany). The chondrocytes culture medium contains Chondrocyte Basal Medium + 10% Chondrocyte Growth Medium SupplementMix (both from PromoCell GmbH, Heidelberg, Germany) + 1% Penicillin/Streptomycin Solution (Life Technologies Europe BV, NN Bleiswijk, Netherlands).

Eitner A., Müller S., König C., Wilharm A., Raab R., Hofmann G.O., Kamradt T, & Schaible H.G. (2021). Inhibition of Inducible Nitric Oxide Synthase Prevents IL-1β-Induced Mitochondrial Dysfunction in Human Chondrocytes. International Journal of Molecular Sciences, 22(5), 2477.

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AICAR, A769662 and Compound C Protocol

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AICAR (5-aminoimidazole-4-carboxamide riboside), A769662 and Compound C were purchased from Tocris. All other chemicals were purchased from Sigma-Aldrich Co.

Kim D., Kang1,2 D., Martin E.A., Kim I, & Carroll J.L. (2014). Effects of modulators of AMP-activated protein kinase on TASK-1/3 and intracellular Ca2+ concentration in rat carotid body glomus cells. Respiratory physiology & neurobiology, 195, 19-26.

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Pharmacological Activators of AMPK Pathway

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Cyclohexamide (350 μM, Cat No. 0970), HHT (50 μM, Cat No. 1416), and A769662 (200 μM, Cat No. 3336) were all obtained from Tocris (Minneapolis, MN). AICAR (2mM, Cat No. 10010241) was obtained from Cayman Chemical Company (Ann Arbor, MI). Metformin (200 mg/kg, intraperitoneal (IP) dose) was obtained from LKT labs. The vehicle for CHX, HHT, AICAR, and A769662 used in culture was sterile 1× PBS. The vehicle for IP metformin dosing was sterile 0.9% saline. For a complete list of reagents and sources see Table 1.

Paige C., Mejia G., Dussor G, & Price T. (2018). AMPK activation regulates P-body dynamics in mouse sensory neurons in vitro and in vivo. Neurobiology of Pain, 5, 100026.

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Modulation of Cellular Pathways with Pharmacological Agents

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Cyclohexamide (350 μM, Cat No. 0970), HHT (50 μM, Cat No. 1416), and A769662 (200 μM, Cat No. 3336) were all obtained from Tocris (Minneapolis, MN). AICAR (2 mM, Cat No. 10010241) was obtained from Cayman Chemical Company (Ann Arbor, MI). Metformin (200 mg/kg, intraperitoneal (IP) dose) was obtained from LKT labs. The vehicle for CHX, HHT, AICAR, and A769662 used in culture was sterile 1X PBS. The vehicle for IP metformin dosing was sterile 0.9% saline. For a complete list of reagents and sources see Table 1.Table 1

Key Reagents List.

Reagent or resource	Concentration used	Source	Identifier
Antibodies
anti-βIII – Tubilin Rabbit	1:1000	Cell Signaling Technology	5568
Anti-Puromycin Mouse	1:5000	Millipore	MABE343
Anti-Rck/p54/p54 Rabbit	1:2000	Cell Signaling Technology	9407
Goat Anti Mouse 546	1:1000	ThermoFisher Scientific	A-11030
Goat Anti Rabbit 488	1:1000	Life Technologies	A11034

Drugs
A769662	200 μM	Tocris	3336
AICAR	2 mM	Cayman Chemical	10010241
Cyclohexamide	350 μM	Tocris	0970
HHT	50 μM	Tocris	1416
Metformin	200 mg/kg	LKT Labs	1115-70-4

Reagents
Puromycin	1 μM	Alomone Labs	P-540
Digitonin	0.00036%	Sigma-Aldrich	D141

Paige C., Mejia G., Dussor G, & Price T. (2018). AMPK activation regulates P-body dynamics in mouse sensory neurons in vitro and in vivo. Neurobiology of Pain, 5, 100026.

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Hedgehog Pathway Activation and Regulation

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Sag was purchased from Adipogen Life Sciences (Liestal, Basel, Switzerland). For Hh pathway activation, DAOY cells were incubated overnight in serum-free medium, containing 1% BSA, and then exposed to Sag (200 nM) for the indicated time points.
AICAR was purchased from Cayman Chemicals, A-769662 from Tocris Bioscience, Metformin and 2-deoxyglucose from Sigma-Aldrich.
DAOY cells were pre-treated with MG132 (Calbiochem, Merk Group) to prevent protein degradation, and then incubated with A-769662. Cells were treated with cycloheximide (CHX, Sigma-Aldrich) and A-769662 as described in the text. The total cell lysates were analyzed by western blot using the indicated antibodies.

Di Magno L., Basile A., Coni S., Manni S., Sdruscia G., D'Amico D., Antonucci L., Infante P., De Smaele E., Cucchi D., Ferretti E., Di Marcotullio L., Screpanti I, & Canettieri G. (2016). The energy sensor AMPK regulates Hedgehog signaling in human cells through a unique Gli1 metabolic checkpoint. Oncotarget, 7(8), 9538-9549.

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Cytotoxic Capacity of NK Cells

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The cytotoxic capacity of NK cells was assessed using the E-Cadherin expressing breast cancer epithelial cell line MCF-10A as target. Briefly, 20,000 MCF-10A cells were plated/well of a 96 flat-bottom plate 24 hours prior to the cytotoxicity assay. On the day of the assay, MCF-10A cells were labeled with Calcein-AM (Sigma-Aldrich) at 10μM for 1 hour before co-culture with NK cells in complete medium containing 500 IU/ml rhIL-2 (Miltenyi Biotech) and as described elsewhere (34 (link)). NK cells transfected with siRNA specific for KLRG1 or scrambled control siRNA for 36 hours were pre-treated with the AMPK agonist A769662 (Tocris Bioscience) at 150 μM or equivalent DMSO as negative control for 2 hours before being added to the target cells. Effector and target cells were combined at a ratio of 40:1 in triplicate and cultured in complete medium containing 500 IU/ml rhIL-2 (Miltenyi Biotech) for 4 hours. After 4 hours of co-culture fluorescence was measured in 75 μl of cell culture supernatant using a Spectramax Gemini spectrofluorimeter. Specific lysis was calculated as % killing = (test release–spontaneous release) / (max release–spontaneous release) x 100.

Müller-Durovic B., Lanna A., Covre L.P., Mills R.S., Henson S.M, & Akbar A.N. (2016). Killer Cell Lectin-like Receptor G1 (KLRG1) inhibits NK cell function through activation of AMP-activated Protein Kinase. Journal of immunology (Baltimore, Md. : 1950), 197(7), 2891-2899.

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Mitochondrial Dysfunction Evaluation Protocol

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NaIO₃ (sodium iodate), N-acetyl cysteine (NAC), trolox, 3-AB (3-aminobenzamide), DPQ (3,4-dihydro-5-[4-(1-piperidinyl) butoxy]-1(2H)-isoquinolinone), DCFDA (dichlorofluorescein diacetate), DHE (dihydroethidium), oligomycin, FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone), rotenone, antimycin A, and U0126 were obtained from Sigma-Aldrich Co (St Louis, MO, USA). MitoSOX and Mitotracker green were purchased from Thermofischer scientific (Waltham, MA, USA). MitoPY1 and A769662 were obtained from Tocris Biosciences (Bristol, UK). Metformin was purchased from Medchem express (Monmouth Junction, NJ, USA). The antibodies specific for phospho-ERK1/2 (T202/Y204), ERK1/2, phosphor-Akt (Ser 473), Akt, phospho-dynamin-related protein (DRP)-1 (S616), DRP-1, γH2AX and Tom 20 were purchased from Cell Signaling Technology (Beverly, MA, USA). The β-actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F12), penicillin, streptomycin and trypsin-EDTA were from Invitrogen (Rockville, MD, USA). The ECL reagent (Western blotting lightening chemiluminescence reagent plus) was purchased from PerkinElmer (Wellesley, MA, USA).

Chan C.M., Sekar P., Huang D.Y., Hsu S.H, & Lin W.W. (2020). Different Effects of Metformin and A769662 on Sodium Iodate-Induced Cytotoxicity in Retinal Pigment Epithelial Cells: Distinct Actions on Mitochondrial Fission and Respiration. Antioxidants, 9(11), 1057.

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