3T3-L1 adipocytes and C2C12 myotubes were treated with metformin (Sangon Bio-Tech, Shanghai, China) as the positive control, phloretin (BioVision, Milpitas, CA, United States) as the negative control, and NAR for 4 h. Glucose uptake was measured using the 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-glucose (2-NBDG) glucose uptake assay kit (BioVision, Milpitas, CA, United States) (Waters et al., 2019 (link)). Images were taken by fluorescence microscope (Zeiss, Jena, Germany). The fluorescence intensity of each photograph was measured by Image-Pro Plus software (Media Cybernetics, Rockville, MD, United States). The drug-free group was used as a control. The relative ratio of each group was obtained after normalization.
Metformin
Manufactured by Sangon
Sourced in China
Metformin is a white to off-white, crystalline powder used as a chemical standard and reference material in analytical procedures. It is a widely used medication for the treatment of type 2 diabetes, acting as an antihyperglycemic agent by reducing hepatic glucose production and increasing glucose uptake in peripheral tissues.
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Lab products found in correlation
Image pro plus software, by Media Cybernetics (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Cck 8 assay, by Dojindo Laboratories (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Control sirna sinc, by GenePharma (1 mentions)
Cyclin d1 92g2, by Cell Signaling Technology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Butyric acid, by Thermo Fisher Scientific (1 mentions)
Dextran standard, by Merck Group (1 mentions)
Deae cellulose 52, by Yuanye Bio-Technology (1 mentions)
Acetic acid, by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Propionic acid, by Thermo Fisher Scientific (1 mentions)
Valeric acid, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay ripa buffer, by Sangon (1 mentions)
Tsc1 sirna, by GenePharma (1 mentions)
Dorsomorphin, by Selleck Chemicals (1 mentions)
Rotenone, by Merck Group (1 mentions)
Glutamax 1, by Merck Group (1 mentions)
11 protocols using metformin
1
Glucose Uptake Assay in Adipocytes and Myotubes
2
Metformin Cytotoxicity in QBC939 Cells
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Twenty-four hours after transfection, 5000 QBC939 cells per well were seeded in 96-well plates. The parental cells were plated onto 96-well plates and treated with metformin (D6198; Sangon) at different concentrations (0, 10, 20, and 50 mM). At the indicated times, a CCK8 assay (Dojindo) was performed to assess the final results. This experiment was performed in triplicate.
3
Nrf2 and SIRT1 Regulation in Cell Stress
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EGCG, ECG and EGC were purchased from Aladdin Chemical (Shanghai, China). Bovine serum albumin (BSA) and methylthiazolyldiphenyl-tetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO). Metformin was purchased from Sangon Biotech (Shanghai, China). Antibodies against Nrf2, Ki-67, PARP-1 (poly(ADP-ribose) polymerase 1), PCNA (proliferating cell nuclear antigen) and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against HO-1, NF-kB p65 (phospho S536) and Caspase-3 (p-17) were purchased from Abcam Inc. (Cambridge, MA). Antibodies against SIRT1 and NF-kB p65 (RELA) were purchased from Proteintech (Rosemont, IL). The pan-acetyllysine antibody was purchased from PTM Biolabs Inc. (Hangzhou, China).
The Nrf2-overexpressing adenovirus (Ad-Nrf2), HO-1-overexpressing adenovirus (Ad-HO-1) and control adenovirus (Ad-NC) were designed and constructed by GeneChem (Shanghai, China). The SIRT1 siRNA (siSIRT1) and control siRNA (siNC) were purchased from GenePharma Co., Ltd. (Shanghai, China).
The Nrf2-overexpressing adenovirus (Ad-Nrf2), HO-1-overexpressing adenovirus (Ad-HO-1) and control adenovirus (Ad-NC) were designed and constructed by GeneChem (Shanghai, China). The SIRT1 siRNA (siSIRT1) and control siRNA (siNC) were purchased from GenePharma Co., Ltd. (Shanghai, China).
4
Investigating Intestinal Cancer in APC^Min/+ Mice
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4- to 5-week-old C57BL/6-APCMin/+ mice, used extensively for intestinal cancer studies, were purchased from Shanghai Model Organisms Center and housed in specific pathogen-free (SPF) and barrier conditions with controlled temperature (20–26 °C) and humidity (40–70%). After 1-week period of adaptive breeding, all mice were randomly divided into 4 groups: negative control (NC), F. nucleatum (FN), F. nucleatum plus Metformin (FM), and Metformin (MET). F. nucleatum was administrated at 109 colony forming units (CFU) suspended with PBS twice per week via gavage. Metformin (Sangon Biotech, China) was fed at a dose of 250 mg/kg by oral gavage every day for 12 weeks. PBS was acted as the control treatment. All mice were sacrificed at 18 weeks. The whole intestinal tract was removed and longitudinally opened to estimate tumour progression. Tumour numbers and sizes (diameter) were carefully recorded. The mouse experiment was conducted according to guidelines approved by the Institutional Animal Care and Use Committee of Renji Hospital, School of Medicine, Shanghai Jiaotong University.
5
Molecular Signaling Pathway Evaluation
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Metformin was purchased from Sangon Biotechnology (Shanghai, China). Rapamycin was purchased from Gene Operation (MI, USA). Antibodies to phospho-ACC (pACC, Ser79), phospho-AMPKα (pAMPKα, Thr172), phospho-mTOR (p-mTOR, Ser2448), phospho-S6 (pS6, Ser235/236), phospho-4EBP1 (p4EBP1, Thr37/46), and Cyclin D1 (92G2) were purchased from Cell Signaling Technology (Beverly, MA, USA). PTEN (G6), MMP7 (L-17), DCN1 (N-15), and anti-β-actin were from Santa Cruz Biotechnology (Dallas, TX, USA).
6
Isolation and Characterization of Dendrobium officinale
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Dendrobium officinale leaves were collected from Yueqing county (Wenzhou, China) in July of 2020. Metformin (BR, 97%) was obtained from Sangon Biotech Co., Ltd (Shanghai, China). DEAE Cellulose-52 was purchased from Yuanye Bio-Technology Co., Ltd (Shanghai, China) and Sephadex G-200 was purchased from Aladdin Bio-Chem Technology Co., Ltd (Shanghai, China). Dextran standards (5000, 11600, 23800, 48600, 80900, 148000, 273000, 409800, 667800 Da), STZ (≥75% α-anomer basis), glucose (AR, 99%), and insulin (BR, 27U/mg) were all purchased from Sigma-Aldrich (Missouri, USA). Antibodies against GRP43, insulin and glucagon as well as their isotype control antibodies were purchased from Proteintech (Chicago, USA). GRP41 and GAPDH antibodies was purchased from Abcam (Cambridge, USA). Acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid (≥99.5) were purchased from Thermo Fisher Scientific (Waltham, USA).
7
Metformin and Glucose Injection Protocols
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Metformin (Sangon Biotech) was dissolved into distilled water at 0.2 mmol/L. For glucose injection, a 0.3 mM glucose solution was prepared (in water) and autoclaved. The injection procedure for glucose and Metformin was the same. A volume of 0.2-µL solution per fourth instar nymph was injected. Water was used as a control. The injection procedures were identical to those for dsRNA injection described above.
8
Metformin and Dorsomorphin Effects on NRK Cells
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After the NRK cells were seeded at 3 × 105 cells per well in 12-well plates for 24 h, Tsc1 siRNA (25 nM) was transfected for 42 h. The siRNA sequence was 5′-CGCCTTTATGGAATGTACCCTTGTA-3′, purchased from Gene Pharma, China. The cells were then treated with 3 mM metformin (Sangon Biotech) with or without 10 μM dorsomorphin (Selleck Chemicals) for 2 h. The NRK cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Sangon Biotech) containing protease inhibitors on ice for 40 min before centrifugation at 12,500 rpm for 20 min for collection of cell lysates. Equal amounts of protein samples were loaded onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for immunoblotting analysis as we described previously37 (link)–39 (link).
9
Berberine and Metformin Compound Evaluation
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Berberine was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Metformin was purchased from Shanghai Sangon Biotechnology Corporation (Shanghai, China). Both drugs were dissolved in ddH2O, which was used as the vehicle for berberine and Metformin. Compound C was purchased from Merck KGaA (Darmstadt, Germany) and dissolved in DMSO, which was used as the vehicle for Compound C. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and other culture reagents were purchased from Gibco Life Technologies (Grand Island, NY). Rotenone, sodium pyruvate, GlutaMax-1 and other chemical reagents were purchased from Sigma Chemicals (St Louis, MO).
10
High-Fat Diet-Induced Metabolic Disorder
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The high-fat diet (HFD) consists of 15% lard (w/w) and 15% sucrose (w/w), 70% basic diet which was obtained from Zhejiang Academy of Medical Sciences (Hangzhou, China). The ingredients of the high-fat diet and standard chow diet are shown in Supplementary Table S1 . The 4-ME was obtained from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China) with a purity of 99.95% in Supplementary Figure S2 . Insulin and leptin Elisa kits were provided by Shanghai Hengyuan Biological Technology Co., Ltd. (Shanghai, China); ML385 was purchased from Glpbio (Montclair, NJ, USA); metformin (purity more than 98%) and fatty free-bovine serum albumin (BSA) were obtained from Sangon Biotech (Shanghai, China). The following items were purchased from the cited commercial sources: CD36, CPT-1A, FASN, PPAR-α, PPAR-γ, TGF-β, Tubulin, and Lamin B antibodies (Proteintech, Chicago, IL, USA); HIF1α antibody (Invitrogen, Carlsbad, CA, USA); SREBP-1c antibody (Novus Biologicals, Littleton, CO, USA); Nrf2 antibody (Affinity Biologicals, Shanghai, China).
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