The human hypopharyngeal carcinoma cell line (FaDu) was purchased from the American Type Culture Collection (ATCC®, Manassas, VA, USA) and stored in liquid nitrogen until use. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum (FCS), 100 IU/ml penicillin and 100 µg/ml streptomycin at 37℃ in 5% carbon dioxide in air. After three consecutive passages, cells were divided into groups: untreated (control); 25 mmol/l metformin; 50 mmol/l metformin; 75 mmol/l metformin; 100 mmol/l metformin; and 125 mmol/l metformin (Sigma, St Louis, MO, USA).
Metformin
Manufactured by Merck Group
Sourced in United States, Germany, China, United Kingdom, Sao Tome and Principe, Canada, Italy, Japan, France, Macao, Singapore, Switzerland, Mexico
Metformin is a laboratory compound used in research and development applications. It serves as a core compound for further chemical modifications and investigations. Metformin is widely utilized in the pharmaceutical industry and academic research settings.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (69 mentions)
Phenformin, by Merck Group (34 mentions)
Streptozotocin (stz), by Merck Group (31 mentions)
Aicar, by Merck Group (29 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (29 mentions)
Compound c, by Merck Group (29 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (26 mentions)
Rapamycin, by Merck Group (25 mentions)
Insulin, by Merck Group (25 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (23 mentions)
Penicillin, by Thermo Fisher Scientific (22 mentions)
Streptomycin, by Thermo Fisher Scientific (22 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (21 mentions)
Rotenone, by Merck Group (20 mentions)
Cisplatin, by Merck Group (15 mentions)
β actin, by Cell Signaling Technology (14 mentions)
Dexamethasone (dex), by Merck Group (13 mentions)
Oligomycin, by Merck Group (13 mentions)
Trypsin edta, by Thermo Fisher Scientific (11 mentions)
Simvastatin, by Merck Group (11 mentions)
848 protocols using metformin
1
Metformin Inhibits FaDu Cell Growth
2
Metformin's Effects on Chondrocyte and Cartilage Inflammation
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Chondrocytes and cartilage explants were grown in culture medium with 10 ng/ml recombinant interleukin-1β (IL-1β) (R&D Systems, USA) and metformin (1, 10, and 20 mM, Sigma-Aldrich, USA). Chondrocytes and cartilage explants were also cultured in the presence of 10 ng/ml recombinant IL-1β alone. A control sample of chondrocytes and cartilage explants cultured in the absence of metformin and IL-1β was also evaluated. Finally, the effect of the addition of metformin was evaluated in the presence of 10 mM metformin and IL-1β samples, with or without dorsomorphin (10 uM, Sigma-Aldrich, USA), which is an AMPK inhibitor. Dimethyl sulfoxide (DMSO) was used as a vehicle of dorsomorphin. The chondrocytes in each group were respectively treated for 24 h by the corresponding intervention methods, then RNA and protein extraction were performed and the medium was collected. The cartilage explants were treated for 48 h. The medium was collected. All in vitro experiments and assays were repeated three times.
3
Metformin Modulates Osteogenesis and Osteoclastogenesis
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Confluent flasks at passage four were trypsinized and cells were seeded at day -1 in 48 wells plates at 1.5∙104 cells per well for osteoclastogenesis assays or 3∙104 cells per well for osteogenesis assays. At day 0, the start of the experiment, the culture medium was removed from the 48-well plates. For the osteogenesis-assay plates, 0.4 ml mineralisation medium - composed of DMEM, 10% FCI, 1% PSF, 50 μg/ml ascorbic acid (Sigma), 10 nM β-Glycerophosphate (Sigma), and different metformin concentrations (or the solvent volume of sterile water, in the case of 0 mM metformin) were added to the wells. For osteoclastogenesis assays, peripheral blood mononuclear cells (PBMCs) derived from a buffy coat (Sanquin, Amsterdam) were added as source containing osteoclast precursors. ACTA has an agreement with bloodbank Sanquin to use buffy coats for research purposes. metformin (Sigma Aldrich, D150959) was dissolved in demineralized water and added to cultures in a range between 0 and 1.0 mM, according to concentrations used in the literature.
For the osteoclastogenesis-assay plates, 0.2 ml containing 5∙105 PBMCs in culture medium were added to the wells as well as 0.2 ml of metformin-consisting medium. During the 21 days duration of the experiment, culture- and mineralization-medium with the appropriate concentration of metformin was replenished twice a week.
For the osteoclastogenesis-assay plates, 0.2 ml containing 5∙105 PBMCs in culture medium were added to the wells as well as 0.2 ml of metformin-consisting medium. During the 21 days duration of the experiment, culture- and mineralization-medium with the appropriate concentration of metformin was replenished twice a week.
4
Metformin Exposure in Wastewater Treatment
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Metformin (1,1-dimethylbiguanidine hydrochloride; CAS # 1115–70-4) and ethanol (200 proof; CAS # 64–17-5) were purchased from Sigma-Adrich. A 100-mg/L stock solution of Metformin was prepared by adding 50 mg of Metformin to 10 mL of ethanol, stirring until dissolved, and then adding this to 490 mL of Milli-Q ultrapure water (EMD Millipore). A final Metformin concentration of 40 µg/L, the average concentration found by our previous study in WWTP effluent [7 (link)], was achieved by adding 8 mL of this stock solution to 20 L of dechlorinated water in each treatment tank. A stock solution for control tanks was prepared by adding 10 mL of ethanol to 490 mL of ultrapure water; then 8 mL of this vehicle stock was added to 20 L of dechlorinated water in each control tank.
5
Metformin Regulation of TRPA1 in Bladder Cells
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In vitro cell experiments were carried out in Primaria plates (BD Falcon) for uroepithelial cells, TERT-NHUC, and polystyrene (Costar) for 5637 and THP1 differentiated cells. Nearly confluent cells, 70–80%, were treated with freshly prepared metformin (4 mM, Sigma-Aldrich) for 24 h, deionized water served as vehicle control. 4 mM metformin concentration was found to be optimal for both the cell lines based on the dose response expression profiles of CAMP and RNASE7 expression (Fig. S1 ).
5' adenosine monophosphate-activated protein kinase (AMPK) activator 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR, 1 mM, Sigma-Aldrich) or AMPK inhibitor, Compound C (20 µM, Sigma-Aldrich) were used and cells were treated for 24 h. In blocking experiments, Compound C was added 1 h prior to adding AICAR or metformin. Chinese hamster ovary cell line expressed growth/differentiation factor 15 peptide (GDF15, 50 ng/ml, R&D systems) was treated to the cells for 24 h. For TRPA1 RT-PCR or imaging experiments, cells were stimulated with the activator, allyl isothiocyanate (AITC, 10 µM, Sigma-Aldrich) and inhibitor A-967079 (A96, 20 µM, Sigma-Aldrich), for 24 h for mRNA analysis. Equal amount of DMSO served as vehicle in control cells.
5' adenosine monophosphate-activated protein kinase (AMPK) activator 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR, 1 mM, Sigma-Aldrich) or AMPK inhibitor, Compound C (20 µM, Sigma-Aldrich) were used and cells were treated for 24 h. In blocking experiments, Compound C was added 1 h prior to adding AICAR or metformin. Chinese hamster ovary cell line expressed growth/differentiation factor 15 peptide (GDF15, 50 ng/ml, R&D systems) was treated to the cells for 24 h. For TRPA1 RT-PCR or imaging experiments, cells were stimulated with the activator, allyl isothiocyanate (AITC, 10 µM, Sigma-Aldrich) and inhibitor A-967079 (A96, 20 µM, Sigma-Aldrich), for 24 h for mRNA analysis. Equal amount of DMSO served as vehicle in control cells.
6
Embryonic Drug Exposure Assay
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Metformin, rapamycin, casticin, and hydroxyurea were bought from Sigma-Aldrich (USA).
Metformin and hydroxyurea were dissolved in water, while the others were dissolved in ethanol. Serial dilutions of each chemical were added to M9 right after the embryos were prepared, so the worms were hatched in a drug treatment environment and influenced by the chemical prior to L1 arrest.
Metformin and hydroxyurea were dissolved in water, while the others were dissolved in ethanol. Serial dilutions of each chemical were added to M9 right after the embryos were prepared, so the worms were hatched in a drug treatment environment and influenced by the chemical prior to L1 arrest.
7
HepG2 Cell Lipotoxicity Modulation
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Human hepatoma HepG2 cells were purchased from the Korean Cell Line Bank (KCLB, Korea) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin, and streptomycin (Gibco, USA) in a humidified atmosphere under 5% CO2 at 37°C. HepG2 cells were seeded in 96-well plates. After reaching confluence, the cells were serum-starved overnight and exposed to 0.4 mM PA (Sigma-Aldrich, USA), in the presence or absence of 5 μM simvastatin (Sigma-Aldrich), 2 mM metformin (Sigma-Aldrich), 50 μM of hesperidin (LKT Laboratories, USA), narirutin (Sigma-Aldrich), nobiletin (Sigma-Aldrich), sinensetin (Sigma-Aldrich), or tangeretin (Sigma-Aldrich) for 24 h. The cells treated with simvastatin or metformin were used as the positive control.
8
Metformin Effects on Adipose Tissue
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Whole adipose tissue was collected at the time of bariatric surgery, weighed and immediately divided into 150 mg pieces under sterile conditions. Samples were incubated at 37 °C 5% CO2 with 3 ml DMEM (Gibco; Cat#4196S-039) enriched with 10% FBS and 1% penicillin-streptomycin (Sigma–Aldrich; Cat# P4333). For metformin-treated explants, the culture media was supplemented with metformin (Sigma–Aldrich; Cat#317240) reconstituted in water at indicated concentrations. Explants were incubated for 48 h and harvested for SVF isolation (measurement of the metformin effect on cell viability) or for UPLC-MS/MS.
9
Obesity, Weight Loss, and Influenza
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Wildtype control and DIO male mice were purchased and acclimated for 2–3 weeks in our animal facility. Following 12 weeks on HFD, obese mice either remained untreated, were switched to normal chow to induce weight loss, or were treated with 250 mg/kg of metformin (Sigma-Aldrich) in drinking water (mouse weight and water consumption were monitored 3 times per week and metformin dose was adjusted accordingly). Following 6 weeks of either weight loss or metformin, mice were lightly anesthetized with isoflurane and infected intranasally with 0.005 HAU per 50μL (TCID50 2.39 ×107) PBS of the H1N1 influenza virus A/Puerto Rico/8/34 (PR8, American Type Culture Collection, Manassas, VA). Body weight was monitored daily following infection. Mice reaching more than 30% body weight loss or other terminal experimental endpoints, such as impaired ambulation or respiratory status, were euthanized by CO2 inhalation.
10
Metformin treatment in male chickens
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Male chickens (Ross) were purchased from the 'Institut de Sélection Animale', Saint Brieuc, France. Chickens were housed with access to feed and water ad libitum and were maintained in a 16 h light:8 h darkness photoperiod. All animal procedures were carried out in accordance with the European legislation for animal experimentation (directive 86/609/EEC) and with French legislation on animal research. The procedures were approved by the Ethics Committee of Val de Loire (CEEA VdL, Comité d'Ethique pour l'Expérimentation Animale du Val de Loire, no. 01607.02). For in vivo exposure, chickens were treated with 0.5 mg/mL metformin in the drinking water (Sigma) starting at 6 weeks of age for 3 weeks in order to provide an average dose of 150 mg/kg per day, with the concentration of metformin adjusted in the drinking water each day (Chen et al. 2011) (link). We carried out two experiments with four birds per condition.
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