All i.p. injections were performed at the same time of day by the same experimenter who was blind to genotypes. PF-4708671 was obtained from Sigma, MO, USA. Littermate matched mice underwent 10 days of i.p injections of 25 mg/kg PF-4708671 in a vehicle consisting of saline + 5% v/v of Tween-80 (Sigma, USA) as previously described16 (link). Lithium citrate (Sigma, USA) in saline or saline was injected at 300 mg/kg ip for 5 days per a previously established paradigm27 (link). Metformin (Tocris, USA) was prepared in saline and injected at 200 mg/kg and administered for 10 days i.p. at 200 mg/kg while saline vehicle only was administered to littermates28 (link). On the last day of treatment, mice were killed by cervical dislocation and the hippocampus was quickly dissected to yield whole hippocampal lysates. Simultaneously, trunk blood also was collected to monitor peripheral levels of proteins. Changes in phosphorylation in hippocampal lysates from treated animals were used to verify drug efficacy for PF (S6), Lithium (GSK-3B), Metformin (ERK). All drug treatments were administered on 2–3 cohorts of mice, some of which were months apart. To combat antibody variability over time, all blots were run with a WT vehicle from that cohort and then protein of interest signal was normalized to the average WT vehicle signal for each blot before being combined and analyzed together.
Metformin
Manufactured by Bio-Techne
Sourced in United States, United Kingdom
Metformin is a laboratory product manufactured by Bio-Techne. It is a chemical compound used in research applications. Metformin is a widely used and studied pharmaceutical agent, but its specific function and intended use are outside the scope of this factual product description.
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Lab products found in correlation
Aicar, by Bio-Techne (3 mentions)
Insulin, by Merck Group (2 mentions)
Metformin, by Merck Group (2 mentions)
Pf 4708671, by Merck Group (1 mentions)
Tween 80, by Merck Group (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Anti akt 9272, by Cell Signaling Technology (1 mentions)
Anti parp 9542, by Cell Signaling Technology (1 mentions)
Rotenone, by Merck Group (1 mentions)
Paclitaxel, by Merck Group (1 mentions)
Puromycin, by Merck Group (1 mentions)
Cholera toxin, by Merck Group (1 mentions)
C57bl 6 mice, by Jackson ImmunoResearch (1 mentions)
Oxamate, by Merck Group (1 mentions)
Hydroxyurea, by Thermo Fisher Scientific (1 mentions)
Chloroquine, by Merck Group (1 mentions)
6 aminonicotinamide, by Merck Group (1 mentions)
Gsk2837808a, by Merck Group (1 mentions)
Th588, by Selleck Chemicals (1 mentions)
Piperlongumine, by Merck Group (1 mentions)
14 protocols using metformin
1
Pharmacological Interventions in Hippocampus
2
Optimization of SCD Inhibitor Studies
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SCD inhibitors were purchased from Biovision and Caymen chemicals. Anti-SCD antibodies (SCD11-A) were from Alpha Diagnostics International and anti-Akt (9272), anti-cytochrome C (11940S) and anti-PARP (9542) from Cell Signalling. Hydrocortisone, EGF, Akt inhibitor (Akt V) and staurosporin were from Calbiochem. Insulin, cholera toxin, puromycin, doxycycline, paclitaxel and rotenone were from Sigma-Aldrich and metformin from Tocris Biosciences.
3
Murine Model of Arthritis Induction
4
Preparation of Compound Stock Solutions
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Solid stocks were purchased from the indicated suppliers and prepared as concentrated stock solutions in the appropriate solvent: hydroxyurea (100 mM in dH2O) from Acros, 2-deoxygluosce (1 M in dH2O), oxamate (100 mM in dH2O), 6-aminonicotinamide (2.5 mM in DMSO), piperlongumine (20 mM in DMSO), simvastin (20 mM in DMSO), L-buthionine-sulfoxamine (50 mM in dH2O) or chloroquine (20 mM in dH2O) from Sigma; GSK 2837808A (10 mM in DMSO) and metformin (100 mM in DMEM) from TOCRIS bioscience and TH588 (10 mM in DMSO) from Selleckchem. V158411 and VER-246008 were from Vernalis Research and prepared as 20 mM DMSO stocks. Compounds were serially diluted in the appropriate solvent to 500× or 1000× final concentration then to 5× or 10× final concentration in complete media before addition to cells to yield a 1× final concentration.
5
Characterization of pNET Cell Lines
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QGP-1, a human pNET cell line, was purchased from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan). NIT-1, a mouse pNET cell line, was purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). The passages of the two cell lines used in this study were less than 15. We sent the QGP-1 cell line to the Center for Genomic Medicine of National Cheng Kung University for genotyping in June 2016, and the result showed the same STR PCR DNA profile as those in the JCRB database. QGP-1 and NIT-1 cells were cultured in RPMI-1640 (HyClone, South Logan, Utah, USA) medium and F12-Kaighn's (Gibco, Grand Island, NY, USA) medium, respectively, containing 10% fetal calf serum and antibiotics. PTEN and c-Myc expression plasmids were purchased from Addgene (Cambridge, MA, USA). shRNAs targeting PTEN, LKB1, AKT and c-Myc were obtained from the National RNAi Core Facility of Academic Sinica (Taipei, Taiwan). Rapamycin and RAD001 were purchased from LC Laboratories (Boston, MA, USA) and Selleckchem (Houston, TX, USA), respectively. Metformin was purchased from TOCRIS (Bristol, UK), and 10058-F4 was purchased from Calbiochem (San Diego, CA, USA).
6
Metformin Treatment on Endometrial Cancer
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RL95‐2 was purchased from ATCC (Manassas, VA, CRL‐1671) and maintained in DMEM:F‐12 medium (ATCC 30‐2006) supplemented with 10% fetal bovine serum (ATCC 30‐2020), 100 U/mL penicillin/100 µg/mL streptomycin (ATCC ATCC 30‐2300), and 0.005 mg/mL insulin (Sigma‐Aldrich, I9278). ACI‐181 (RRID:CVCL_N828),18 a model of endometrioid endometrial cancer (EEC) was a gift from Dr John Risinger (Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University) and cultured in DMEM:F12 supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. Both cell lines were maintained at 37°C and 5% CO2. Metformin was purchased from Tocris (2864). For the Metformin time course experiments, 100 000 and 200 000 of ACI‐181 and RL95‐2 were seeded in 6‐well plates and treated with 20 mmol/L of Metformin for 96 and 120 hours. Two independent experiments were performed with one biological sample for each treatment condition per cell line.
7
Metformin and AICAR Effects on Blood Pressure
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After the recovery period, blood pressure measurements were obtained continuously for the duration of the experiment. Plotted mean arterial pressure (MAP) data represent mean measurements from 9:00 a.m. to 12:00 p.m. as described previously (Pavlov et al., 2013 (link)). 20 mg/kg/day of AICAR or 200 mg/kg/day metformin (Tocris, Minneapolis, MN) administration was performed with continuous infusion at a rate of 6.9 μL/min through the venous catheter in saline, which was also used as a vehicle. After 3 days recovery when animals were fed a normal 0.4% NaCl diet, rats were switched to a 4 or 8% NaCl diet for 3 weeks (Dyets, Bethlehem, PA). Urine samples were collected during infusion experiments and Na+, K+, protein, and creatinine levels were analyzed as previously described (Pavlov et al., 2016 (link)). Blood was collected during the experiment with bleeds from the arterial catheters on conscious rats while resting, and plasma glucose level was then measured with a Contour strip glucometer (Bayer, Mishawaka, IN). At the end of the protocols, the kidneys of rats were perfused (6 ml/min) through the distal aorta with saline to clear them from blood, and then the renal tissues were collected for further analyses (Pavlov et al., 2015a (link)).
8
Oral antidiabetic drugs and neuropathic pain
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The effects of oral linagliptin (3 mgkg−1; a kind gift from Boehringer Ingelheim Pharmaceuticals, Ingelheim, Germany) and metformin (200 mgkg−1; Sigma Aldrich, Poole, UK) on the development of HFD/STZ induced changes in mechanical hindpaw withdrawal thresholds were quantified, with the drugs administered from day 4 until day 40 and compared to saline vehicle (HFD/Veh/saline: n = 8; HFD/STZ/saline: n = 8; HFD/STZ/linagliptin: n = 7; HFD/STZ/metformin: n = 6).
The effects of oral daily pioglitazone (10 mgkg−1, in 1% methylcellulose; Tocris Cookson, Bristol, UK) versus vehicle on the development of HFD/STZ induced changes in mechanical hindpaw withdrawal thresholds were quantified once diabetes was established, from day 21 to day 49 after STZ treatment (n = 10-11 per group).
The dose of 3 mgkg−1 of linagliptin was used as this dose has been reported to improve glucose control when given once daily in animal models of diabetes [26 (link)]. In addition this dose significantly increased plasma GLP-1 in diet-induced obese rats and mice [27 (link), 28 (link)] and caused 67–80% DPP-4 inhibition [28 (link)]. The doses of 200 mgkg−1 of metformin and 10 mgkg−1 of pioglitazone were selected as these doses prevent the development of, or reverse established, pain hypersensitivity when given orally in rats [29 (link)–32 (link)].
The effects of oral daily pioglitazone (10 mgkg−1, in 1% methylcellulose; Tocris Cookson, Bristol, UK) versus vehicle on the development of HFD/STZ induced changes in mechanical hindpaw withdrawal thresholds were quantified once diabetes was established, from day 21 to day 49 after STZ treatment (n = 10-11 per group).
The dose of 3 mgkg−1 of linagliptin was used as this dose has been reported to improve glucose control when given once daily in animal models of diabetes [26 (link)]. In addition this dose significantly increased plasma GLP-1 in diet-induced obese rats and mice [27 (link), 28 (link)] and caused 67–80% DPP-4 inhibition [28 (link)]. The doses of 200 mgkg−1 of metformin and 10 mgkg−1 of pioglitazone were selected as these doses prevent the development of, or reverse established, pain hypersensitivity when given orally in rats [29 (link)–32 (link)].
9
Antibodies for AMPK and Tau Signaling
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Antibodies directed against AMPKα1/α2, ACC, pSer79-ACC were obtained from Cell Signaling Technology. Anti-AMPK α1 and anti-AMPK α2 antibodies were from R&D Systems. Anti p-Thr172AMPK antibody and p-Thr172AMPK blocking peptide were obtained from Santa Cruz Biotechnology24 (link). Anti-actin antibody was from BD Transduction Laboratory. Anti-MAP2, anti-α-tubulin and anti-acetylated-α-tubulin antibodies were from Sigma. Mouse monoclonal antibodies PHF1 (tau pSer396/Ser404)50 (link), CP13 (tau pSer202)51 (link), 2E12 or RZ3 (tau pThr231)5 (link), DA9 (total tau aa102-140)36 (link), DA31 (total tau aa220-240)36 (link) and MC1 (conformation dependent antibody that recognizes only tau in a pathological conformation)52 (link) were previously described. 12E8 monoclonal antibody was obtained from Dr. Seubert (Elan Pharmaceuticals, San Francisco, CA; tau pSer262/356)53 (link). As total tau antibody in the Western-blot experiments, we used tau Cter (homemade well characterized antibody recognizing the 11 amino-acids in the c-terminal part of tau27)54 (link). Tau5 (total tau) and AT180 (tau pThr231) were from Invitrogen.
AICAR, metformin, PD98059, H-89, LY294002 were purchased from Tocris; roscovitin and MARK/Par1 inhibitor from Merck; Compound C was from Santa-Cruz Biotechnology and rapamycin was from Cell Signaling Technology.
AICAR, metformin, PD98059, H-89, LY294002 were purchased from Tocris; roscovitin and MARK/Par1 inhibitor from Merck; Compound C was from Santa-Cruz Biotechnology and rapamycin was from Cell Signaling Technology.
10
Metabolic Signaling Pathway Modulators
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Rotenone, AICAR (5-aminoimidazole-4-carboxamide riboside), A-769662, metformin, ZLN024 and PT1 were purchased from Tocris and 2-deoxy-D-glucose (2DG) was purchased from Abcam. Oligomycin and antibodies to MLC, pMLC, pHSP27, pAMPKα, pACC were purchased from Cell Signaling Technology. Salicylate was purchased from Sigma. The anti-VE-cadherin and ROKα antibodies were from BD-Biosciences and the anti-RhoA antibody was from Merck Millipore. Glucose-free DMEM and dialysed serum were purchased from Gibco Life Technologies (Paisley, United Kingdom).
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