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Sto 609

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STO-609 is an inhibitor that targets the calcium/calmodulin-dependent protein kinase kinase (CaMKK) enzyme. It is intended for use in research applications.

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Lab products found in correlation

Kn 93, by Bio-Techne (4 mentions) Pluronic f 127, by Thermo Fisher Scientific (1 mentions) Bapta am, by Thermo Fisher Scientific (1 mentions) Egta am, by Thermo Fisher Scientific (1 mentions) Okadaic acid, by Bio-Techne (1 mentions) Nimodipine, by Abcam (1 mentions) Sterile dimethylsulfoxide, by Merck Group (1 mentions) Wnt5a, by R&D Systems (1 mentions) Anti β actin, by Merck Group (1 mentions) Hrp conjugated secondary ab, by Jackson ImmunoResearch (1 mentions) Anti iκb, by Cell Signaling Technology (1 mentions) Cp690550, by Bio-Techne (1 mentions) Recombinant mouse il 10, by R&D Systems (1 mentions) Anti nf κb, by Cell Signaling Technology (1 mentions) Anti phospho nf κb, by Cell Signaling Technology (1 mentions) Gateway cloning, by Thermo Fisher Scientific (1 mentions) Cdcl2, by Merck Group (1 mentions) Actinomycin d, by Merck Group (1 mentions) Cycloheximide (chx), by Cell Signaling Technology (1 mentions) Triptolide, by Bio-Techne (1 mentions)

Close spelling variants of this product

STO-609 acetate (5 citations)

21 protocols using sto 609

1

Inducing CREB Phosphorylation in Cortical Neurons

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To induce CREB phosphorylation, we stimulated cortical neurons with the indicated high [K+] solution at 37 °C for 10–300 s, and fixed the cells immediately after the stimulation (in 4% paraformaldehyde in PBS, with 20 mM EGTA and 4% (w/v) sucrose), or incubated the cells in culture media for 90 min at 37 °C before fixation. Where indicated, drugs were added 30 min before and included throughout the stimulation. All K+-rich stimulation solutions contained 0.5 μM TTX (Ascent Scientific) to block action potentials. In addition, when stimulating cortical neurons, 10 μM NBQX (Ascent Scientific) and 10 μM APV (Ascent Scientific) were included to block AMPA and NMDA receptors, respectively. 4 mM K Tyrode’s consisted of (in mM): 150 NaCl, 4 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, 10 glucose, pH 7.4. When stimulating with elevated [K+], Na+ was adjusted to maintain osmolarity. To block: CaM Kinases, KN93 (Tocris) was used at 4uM; to block CaV1 channels, Nimodipine (abcam) was used at 10uM; to block CaMKK, STO609 (Tocris) was used at 3.3uM; to block PP2A, okadaic acid (Tocris) was used at 20 nM; to block PP1 and PP2A, okadaic acid (Tocris) was used at 2uM.
Cells were loaded with Ca2+ chelators EGTA-AM and BAPTA-AM (Life Technologies), used at 200uM, along with 1:1000 Pluronic F-127 (Life Technologies) 45 min before stimulation.
Cohen S.M., Li B., Tsien R.W, & Ma H. (2015). Evolutionary and functional perspectives on signaling from neuronal surface to nucleus. Biochemical and biophysical research communications, 460(1), 88-99.
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2

STO-609 and GSKi Treatment for Tumor Growth

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STO-609 and GSK1901320 (referred to as GSKi) were synthesized at the Duke University Small Molecule Synthesis Facility. These drugs were administered daily by intraperitoneal injection (10–100 μmoles kg−1 body weight). Sterile dimethyl sulfoxide (Sigma, D2438) was used as a vehicle. STO-609 treatment was initiated at post-graft day 1 for E0771, day 7 for Met1, and day 2 for 4T1 cells. The E0771 tumor growth experiment was repeated independently by a different investigator using a different batch of STO-609 (Tocris Biosciences, Bristol, UK). Treatment with GSKi was initiated at post-graft day 2.
Racioppi L., Nelson E.R., Huang W., Mukherjee D., Lawrence S.A., Lento W., Masci A.M., Jiao Y., Park S., York B., Liu Y., Baek A.E., Drewry D.H., Zuercher W.J., Bertani F.R., Businaro L., Geradts J., Hall A., Means A.R., Chao N., Chang C.Y, & McDonnell D.P. (2019). CaMKK2 in myeloid cells is a key regulator of the immune-suppressive microenvironment in breast cancer. Nature Communications, 10, 2450.
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3

Morphometric Analysis of Cortical Neurons

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Wnt5a (R&D Systems), KN-93 (Calbiochem), or STO-609 (Tocris Bioscience) were applied to the medium of cultured cortical neurons expressing mRFP1 for morphometric analysis from 6 h after plating onwards [21 (link)]. Final drug concentrations were 400 ng/ml (Wnt5a), 10 μM (KN-93), or 2.6 μM (STO-609). Bath application was performed by dissolving the reagents in one-half volume of the conditioned culture medium and then mixing this volume gently with the remaining volume of the original medium that remained in the dish. The culture medium was not changed before fixation.
Horigane S.I., Ageta-Ishihara N., Kamijo S., Fujii H., Okamura M., Kinoshita M., Takemoto-Kimura S, & Bito H. (2016). Facilitation of axon outgrowth via a Wnt5a-CaMKK-CaMKIα pathway during neuronal polarization. Molecular Brain, 9, 8.
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4

In vivo Evaluation of CaMKK2 Inhibitor

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STO-609 was purchased from TOCRIS Bioscience (Ellsville, MO, USA) and prepared as mentioned previously [19 (link)]. Tri-weekly intraperitoneal (i.p.) injections of 200 μl per mouse of either saline (n=9) or 10 μM STO-609 (n=9) were administered into 32 week old WT mice for 6 weeks. Seven and two days before euthanasia, the saline (n=5) and STO-609 treated mice (n=8) were injected via i.p. with calcein (5 mg/ml) and alizarin red (15 mg/ml) respectively, at 100 μl per mouse. Long bones were harvested; tibiae were utilized for dynamic histomorphometry and femurs were utilized for microcomputed tomography (μCT) and mechanical testing. Microarchitecture analyses as well as assessment of both cortical and trabecular bone strength were performed. Untreated 12 week old WT (n=11) and Camkk2−/− (n=11) mice as well as 32 week old Camkk2−/− mice (n=6) were used as controls for μCT and strength analyses.
Pritchard Z.J., Cary R.L., Yang C., Novack D.V., Voor M.J, & Sankar U. (2015). Inhibition of CaMKK2 Reverses Age-Associated Decline in Bone Mass. Bone, 75, 120-127.
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5

Interleukin-10 Signaling Pathway Analysis

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Recombinant mouse IL-10 was purchased from R&D Systems. LY294002 and rapamycin were purchased from Calbiochem (EMD Millipore). CP-690550 and STO-609 were purchased from Tocris Biosciences. Western blot detection of specific proteins used the following primary Abs: anti-phospho-AMPKα (Thr172), anti-AMPKα, anti-phospho-PI3 kinase p85 (Tyr458)/p55 (Tyr199), anti-PI3 kinase p55, anti-PI3 kinase p85, anti-phospho-Akt (Ser473), anti-phospho-Akt (Thr308), anti-Akt, anti-phospho-mTOR (Ser2448), anti-mTOR, anti-phospho-p70 S6K (Ser371), anti-p70 S6K, anti-phospho-TSC2 (Ser939), anti-phospho-TSC2 (Ser1387), anti-TSC2, anti-phospho-Stat3 (Tyr705), anti-phospho-Stat3 (Ser727), anti-Stat3, anti-phospho-JAK1 (Tyr1022/1023), anti-JAK1, anti-SOCS3, anti-phospho-NF-κB (Ser536), anti-NF-κB, anti-IκB (Cell Signaling Technology), anti-β-actin (Sigma-Aldrich), and HRP-conjugated secondary Ab (Jackson ImmunoResearch Laboratories).
Zhu Y.P., Brown J.R., Sag D., Zhang L, & Suttles J. (2014). AMP-activated protein kinase regulates IL-10-mediated antiinflammatory signaling pathways in macrophages. Journal of immunology (Baltimore, Md. : 1950), 194(2), 584-594.
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6

Constructs and Knockdown of β-PIX and Leptin Receptor

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Constructs expressing tagged proteins were constructed by amplifying β-PIX and long isoform of leptin receptor from rat cDNA and cloned into pCAGGS destination vectors containing the designated tags using Gateway cloning (ThermoFisher). The short hairpin RNAs (shRNA) targeting β-PIX (5’-GTTCGATACGACTGCCATCAA-3’) and leptin receptor (5’-GCTCACTGTCTGTTCAGTGAC-3’) were used as previously described [31 (link), 34 (link)]. Full-length rat recombinant leptin (50nM, Peprotech #400-21) and STO-609 (20μM, Tocris #1551) were used as described in the text and figure legends. Mouse LepRb construct was used as shRNA-resistant LepRb, as shLepRb only targets rat LepRb mRNA. The mutant LepRb constructs LepRY985L and LepRY1138S were generous gifts from Martin Myers (University of Michigan). shRNA-resistant β-PIX was achieved by mutating 3 nucleotides using Q5 site-directed mutagenesis kit (NEB) according to manufacturer’s protocol. SH3 domain of β-PIX was deleted using Q5 DNA Polymerase (NEB) following manufacturer’s protocol.
Sahin G.S., Rodriguez-Llamas J.L., Dillon C., Medina I., Appleyard S.M., Gaiarsa J.L, & Wayman G.A. (2021). Leptin increases GABAergic synaptogenesis through the Rho guanine exchange factor β-PIX in developing hippocampal neurons. Science signaling, 14(683), eabe4111.
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7

Cadmium-Induced Osteoblast Cytotoxicity

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Cells were plated at different densities depending on the assay. After 24 h, treatment was initiated with 0.1–10 μM CdCl2 (Sigma–Aldrich, St. Louis, MO), 5 μM calmodulin-dependent PDE inhibitor CGS-9343β (Santa Cruz Biotechnology, CA, USA), 5 μM or 10 μM CAMKK inhibitor STO-609 (Tocris, Bristol, UK), 2.5 μM CAMKII inhibitor KN-93 (Tocris, Bristol, UK), or a co-treatment of CdCl2 and inhibitor for 24 or 48 h (ATCC, Manassas, VA). For the cytotoxicity studies controls received OPTI-MEM serum-free medium only and for inhibitor studies the controls received OPTI-MEM serum-free medium containing 0.01% or less DMSO. A cytotoxicity profile using the MTT assay determined that 5 μM CGS-9343β, 5 μM or 10 μM STO-609, and 2.5 μM KN-93 were not cytotoxic and these concentrations were used for co-treatment experiments (data not shown). The CdCl2 concentrations used are within the concentration range and exposure time reported in the literature (Pulido and Parrish, 2003 (link); Liu and Templeton, 2007 (link); Chen et al., 2011 ) and specifically for osteoblast cultures (Martineau et al., 2010 (link); Arbon et al., 2012 (link); Liu et al., 2014 (link), 2016 ).
Ha T.T., Burwell S.T., Goodwin M.L., Noeker J.A, & Heggland S.J. (2016). Pleiotropic roles of Ca+2/calmodulin-dependent pathways in regulating cadmium-induced toxicity in human osteoblast-like cell lines. Toxicology letters, 260, 18-27.
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8

Calcium-Mediated Neuronal Stimulation Assay

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At 6 or 7 days in vitro (DIV), neurons were silenced with APV (100uM, Tocris) and NBQX (10uM, Tocris) to block NMDA and AMPA receptors. 14-16 hours later neurons were stimulated with a final concentration of 55mM potassium chloride using KCl depolarization solution (170mM KCl, 10mM Hepes pH 7.4, 1mM MgCl2, 2mM CaCl2). For sustained stimulation, KCl was left on neurons for up to 6 hours, whereas for brief stimulation, it was added for one minute, and then removed and replaced with conditioned neurobasal supplemented with APV and NBQX until RNA collection. While sustained KCI-mediated depolarization elevates intracellular calcium for a minimum of 20 minutes and likely indefinitely (Dolmetsch et al., 2001 (link); Evans et al., 2013 (link)), brief KCl-mediated depolarization elevates intracellular calcium only during the period of elevated KCl (Kingsbury et al., 2007 (link)). 10μM U0126 (Tocris), 625nM 11e (Tocris), 3μM STO-609 (Tocris), 30μM cycloheximide (Cell Signaling) or DMSO (equal volume) were added 30 minutes before stimulation and left on the neurons throughout the experiment. 10μg/mL ActinomycinD (Sigma) was added 15 minutes before stimulation. 10μM triptolide (Tocris) was added 5 minutes before stimulation.
Tyssowski K.M., DeStefino N.R., Cho J.H., Dunn C.J., Poston R.G., Carty C.E., Jones R.D., Chang S.M., Romeo P., Wurzelmann M.K., Ward J.M., Andermann M.L., Saha R.N., Dudek S.M, & Gray J.M. (2018). Different neuronal activity patterns induce different gene expression programs. Neuron, 98(3), 530-546.e11.
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9

Oxygen-Glucose Deprivation in Neurons

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At DIV 10–12, neurons were subjected to oxygen–glucose deprivation (OGD) in neurobasal‐A medium lacking glucose and incubated in a hypoxia chamber (Billups‐Rothenburg Inc. Del Mar, CA, USA) filled with an anoxic atmosphere of 5% CO2 balanced in nitrogen at 37°C for 2 h. Cells were incubated with 5‐aminoimidazole‐4‐carboxamide 1‐β‐d‐ribofuranoside a precursor for ZMP, an AMP mimetic (AICAR; 2 mM), the calcium ionophore ionomycin (an activator of CaMKKβ; 2 μM final concentration), or the CaMKKβ inhibitor STO609 (10 μg/mL), all from Tocris Bioscience, Bristol, UK. Pharmacological modulators were incubated with the cells either during OGD (OGD experiments), or for 2 h at 3 h or 24 h prior to OGD (pre‐treatment experiment). Culture medium was assayed for release of lactate dehydrogenase (LDH) (described below), and cell lysates prepared in ice‐cold HEPES Buffer A (50 mM HEPES (pH 7.5), 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM EDTA, +1 × protease inhibitors) containing 1% (v/v) Triton X‐100.
Rousset C.I., Leiper F.C., Kichev A., Gressens P., Carling D., Hagberg H, & Thornton C. (2015). A dual role for AMP‐activated protein kinase (AMPK) during neonatal hypoxic–ischaemic brain injury in mice. Journal of Neurochemistry, 133(2), 242-252.
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10

Mitochondrial Function Optimization

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Etomoxir (ETO) and STO-609 was purchased from TOCRIS (Tocris Bioscience, UK). Bezafibrate (BEZA), CCCP, and H2O2 were obtained from Sigma (St. Louis, MO, USA). Oligomycin was purchased from Cell Signaling Technology (USA). cyclosporin A (CsA) was obtained from Selleck Chemicals (USA) and BAPTA-AM was purchased from MedChemexpress Co. (USA). The calcium-sensitive indicator, Fluo-3 AM was purchased from Beyotime Co. (China). XF Palmitate-BSA FAO Substrate was obtained from seahorse Bioscience.
Wang M.D., Wu H., Huang S., Zhang H.L., Qin C.J., Zhao L.H., Fu G.B., Zhou X., Wang X.M., Tang L., Wen W., Yang W., Tang S.H., Cao D., Guo L.N., Zeng M., Wu M.C., Yan H.X, & Wang H.Y. (2016). HBx regulates fatty acid oxidation to promote hepatocellular carcinoma survival during metabolic stress. Oncotarget, 7(6), 6711-6726.
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