The antibodies used in this study were as follows: anti-P2X7 (Abcam, Cambridge, UK; cat. no. ab109054), anti-collagen II (Abcam; cat. no. ab34712), anti-MMP13 (Abcam; cat. no. ab39012), anti-AMPKα1 (Abcam; cat. no. ab32047), anti-mTOR (Abcam; cat. no. ab109268), anti-NLRP3 (Proteintech; cat. no. 19771-1-AP), anti-caspase-1 (Proteintech; cat. no. 22915-1-AP), anti-LC3B (Abcam; cat. no. ab192890), anti-Beclin-1 (Abcam; cat. no. ab62557), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Proteintech; cat. no. 10494-1-AP), and horseradish peroxidase (HRP)-labeled IgG (Beyotime; cat. no. A0208). The reagents used in the experiment were as follows: MIA (Sigma, St. Louis, MO, USA; cat. no. I2512), the P2X7 receptor agonist BzATP (Sigma; cat. no. B6396), the mTOR activator MHY1485 (Sigma; cat. no. SML0810), the mTOR inhibitor rapamycin (Sigma; cat. no. V900930), the NLRP3 inhibitor CY-09 (Sigma; cat. no. SML2465), the AMPK activator A-769662 (Sigma; cat. no. SML2578), and the AMPK inhibitor compound C (Sigma; cat. no. P5499). The concentrations, dosages, and preparation of the reagents were described previously [43 (link), 45 (link)].
A 769662
Manufactured by Merck Group
Sourced in United States
A-769662 is a chemical compound used in laboratory research. It functions as an activator of the AMP-activated protein kinase (AMPK) enzyme. This compound is commonly utilized in experimental studies to investigate the role of AMPK in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Compound c, by Merck Group (4 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Insulin, by Merck Group (2 mentions)
Sto 609, by Merck Group (2 mentions)
Nucleofector kit, by Lonza (2 mentions)
Human ampkα shrna, by Santa Cruz Biotechnology (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Bafilomycin a1, by Merck Group (2 mentions)
Ml sa1, by Merck Group (2 mentions)
Mk6 83, by Merck Group (2 mentions)
Ml si3, by Merck Group (2 mentions)
Il 15, by Miltenyi Biotec (2 mentions)
Rapamycin, by Merck Group (1 mentions)
Mhy1485, by Merck Group (1 mentions)
Bzatp, by Merck Group (1 mentions)
Anti nlrp3, by Proteintech (1 mentions)
Anti beclin1, by Abcam (1 mentions)
Anti lc3b, by Abcam (1 mentions)
Anti collagen 2, by Abcam (1 mentions)
Anti mmp13, by Abcam (1 mentions)
13 protocols using a 769662
1
Investigating Molecular Pathways in Osteoarthritis
2
Hepatocyte Isolation and Treatment Assay
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Primary hepatocytes were isolated using a two-step collagenase perfusion method as previously described [30 (link)]. Primary hepatocytes were cultured in William’s E medium (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), 2 mM L-glutamine (Sigma, MO, USA), 0.1 μM insulin (Sigma), 0.1 μM dexamethasone (Sigma) and 1% penicillin/streptomycin (Beyotime, Shanghai, China). Primary hepatocytes were subsequently incubated with PbAc (150 μg/mL), MWCNTs (10 μg/mL) or both of them, and intervened with PF-06409577 (0.5 μM), A-769662 (10 μM) or T0070907 (50 μM) for 24 h (compounds were all purchased from Sigma). Sterile saline water or dimethyl sulfoxide (DMSO) was administrated as the control. Finally, the treated primary hepatocytes were used for further apoptotic analysis, western blot analysis and cell viability assay.
3
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.
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.
4
Investigating Transcriptional Regulation in Neuronal Signaling
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Corticosterone, STO609 (CAMKK inhibitor), A769662 (AMPK activator), glutamate, oligomycin, and carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) are from Sigma-Aldrich S.A.R.L. Doxycycline is from Clontech. Brain-derived neurotrophic factor (BDNF) is from Abnova. Tetramethylrhodamine methyl ester (TMRM) is from Santa Cruz Biotechnology. The antibodies used were as follows: cyto-NR4A1 (ABIN460855, antibodies-online.com); pan-NR4A1 (E6, Santa Cruz Biotechnology); and anti-human NR4A1 (D63C5), phospho (p)-NR4A1 (S350-P), ubiquitin (P4D1), P190A, phospho-acetyl-CoA carboxylase (ACC; S79-P), AMPK and phospho-AMPK (T172-P), HDAC2, and S6 (Cell Signaling Technology). Actin is from Sigma-Aldrich S.A.R.L.; FKBP51 and HSP90 are from BD Biosciences-Europe; GAPDH is from Thermo Fisher Scientific; and GFP and Drebrin (M2F6) are from Abcam. RFP is from Rockland Immunochemicals; PSD-95 is from NeuroMab; synaptophysin is from Thermo Fisher Scientific; and NR1 (ab1516) and NR2 (ab1548) are from Merck Millipore.
5
Culturing and Treating Primary Immune Cells
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Human PBMCs were cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Biowest), 100 U penicillin–streptomycin (Gibco), and 2 mM L-Glutamine (Gibco). PDECs were cultured in MammoCult (STEMCELL Technologies), and the MammoCult media was supplemented with MammoCult proliferation supplement #05622 (STEMCELL Technologies), 20 µg/mL gentamicin (Sigma), 0.1 µg/mL amphotericin B (Biowest) and 10,000 U/mL penicillin/streptomycin (Lonza). Cells and PDECs were grown in a humidified incubator at 37°C under 5% CO2, and atmospheric oxygen levels.
PDECs were treated with 25 ul/mL anti-CD3/CD28/CD2 (STEMCELL Technologies), 100 ug/mL atezolizumab (Selleck Chemicals), 50 ug/mL pembrolizumab (MedChem), 10–100 nM Venetoclax (MedChem Express), 5–10 mM metformin (MedChem Express), 10–50 nM paclitaxel (MedChem Express), 1–2.5 nM IACS-010759 (Selleck Chemicals), 10–30 nM rotenone (Sigma-Aldrich), and 10 nM-1 uM A-769662 (Sigma-Aldrich), 100 ng/mL lipopolysaccharide (LPS).
PDECs were treated with 25 ul/mL anti-CD3/CD28/CD2 (STEMCELL Technologies), 100 ug/mL atezolizumab (Selleck Chemicals), 50 ug/mL pembrolizumab (MedChem), 10–100 nM Venetoclax (MedChem Express), 5–10 mM metformin (MedChem Express), 10–50 nM paclitaxel (MedChem Express), 1–2.5 nM IACS-010759 (Selleck Chemicals), 10–30 nM rotenone (Sigma-Aldrich), and 10 nM-1 uM A-769662 (Sigma-Aldrich), 100 ng/mL lipopolysaccharide (LPS).
6
AMPK Activation and Leptin Signaling
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The AMPK activator A769662 and AMPK inhibitor Compound C were purchased from Sigma-Aldrich. Circulating leptin levels were determined using Leptin Human ELISA Kit from Invitrogen. Human recombinant leptin protein was from Thermo Fisher Scientific. Human LepR expression vector and the control were from OriGene, while human AMPKα shRNA and the control were from Santa Cruz Biotechnology. Human CD4 T cells were transfected with electroporation using Nucleofector kits from Lonza. All reagents were used according to the manufacturers’ instructions.
7
Antibodies in Melanogenesis Regulation
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Antibodies against total LKB1(liver kinase B1) (3047), phospho (Serine 428)-LKB1 (3482), total AMPKα (5831), phospho (threonine 172)-AMPKα (2535), total ACC (3662), phospho (serine 79)-ACC (acetyl-CoA carboxylase) (3661), p44/42 MAPK (4695), Phospho-p44/42 MAPK (4376), p38 MAPK (9212), Phospho-p38 MAPK (9211), JNK (9252), Phospho-SAPK/JNK (4668), CREB (9197) and Phospho-CREB (9198) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against tyrosinase related protein 1 (Trp1, sc-166857), tyrosinase related protein 2 (Trp2, sc-74439), tyrosinase (sc-20035), microphthalmia-associated transcription factor (MITF, sc-52938), β-actin (sc-47778), and c-Myc tag (sc-47694) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The antibody against melanocortin 1 receptor (MC1R, PA5-21911) and all cell culture media and supplements were obtained from Thermo Fisher Scientific (Waltham, MA, USA) and Gibco (Grand Island, NY, USA). α-MSH, Arbutin, AICAR, compound C , PD98059, SP600125, SB203580, H89, A-769662, BAPTA-AM and STO-609 were obtained from Sigma-Aldrich (St. Louis, MO, USA).
8
TRPML1 Agonist and Antagonist Assays
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The TRPML1 agonist MK6-83 (Sigma-Aldrich) was dissolved in DMSO and used at 20 μΜ unless specified otherwise. The TRPML1 antagonist ML-SI3 (enamine) was dissolved in DMSO and used at 50 μΜ. The chemical modulators ML-SA1, A-769662, bafilomycin A1, ionomycin, MGR1, and MGR2 were used as indicated in the figure legends, and all were obtained from Sigma-Aldrich. Clinical-grade recombinant human IL-2 (Proleukin) was obtained from Clinigen via Sykehusapoteket (no. 600373). IL-15 was used at 1 or 10 ng/ml (Miltenyi Biotec).
9
TRPML1 Agonist and Antagonist Assays
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The TRPML1 agonist MK6-83 (Sigma-Aldrich) was dissolved in DMSO and used at 20 μΜ unless specified otherwise. The TRPML1 antagonist ML-SI3 (enamine) was dissolved in DMSO and used at 50 μΜ. The chemical modulators ML-SA1, A-769662, bafilomycin A1, ionomycin, MGR1, and MGR2 were used as indicated in the figure legends, and all were obtained from Sigma-Aldrich. Clinical-grade recombinant human IL-2 (Proleukin) was obtained from Clinigen via Sykehusapoteket (no. 600373). IL-15 was used at 1 or 10 ng/ml (Miltenyi Biotec).
10
Assessing Oxidative Stress and Mitochondrial Function
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To detect ROS levels, FLS were stained with CellROX or MitoSOX™ Red mitochondrial superoxide indicator (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s instructions. To determine mitochondrial membrane potentials, TMRM (Tetramethylrhodamine, Methyl Ester, Perchlorate; Invitrogen, Thermo Fisher Scientific) was used according to the manufacturer’s instructions. FLS were analyzed on a FACSCanto2 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and data were processed using FlowJo software v 10.8 (Tree Star, Ashland, OR, USA).
GLX351322 (MedKoo Biosciences, Morrisville, NC, USA) was used to inhibit NOX4 in these cellular processes, and MitoTEMPO (Sigma-Aldrich) was used as a specific scavenger of mitochondrial superoxide. Human recombinant TNF-a (10 ng/mL) and IL-17 (10 ng/mL) were obtained from PeproTech (Cranbury, NJ, USA). A769662 (Sigma-Aldrich) was employed for activating AMPK-mediated cellular signaling.
GLX351322 (MedKoo Biosciences, Morrisville, NC, USA) was used to inhibit NOX4 in these cellular processes, and MitoTEMPO (Sigma-Aldrich) was used as a specific scavenger of mitochondrial superoxide. Human recombinant TNF-a (10 ng/mL) and IL-17 (10 ng/mL) were obtained from PeproTech (Cranbury, NJ, USA). A769662 (Sigma-Aldrich) was employed for activating AMPK-mediated cellular signaling.
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