SH-SY5Y cells were routinely grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO, Gaithersburg, MD, USA) and cultured at 37 °C under humidified 5% CO2 atmosphere. MPP+ (Sigma–Aldrich, St. Louis, MO, USA) and phenformin (Selleckchem, Houston, USA) were freshly dissolved in phosphate buffered saline (PBS) at a stock concentration at 125 mM and 50 mM which was stored at −20 °C. MPP+ and phenformin were further diluted in serum free DMEM to achieve the final concentrations.
Phenformin
Manufactured by Selleck Chemicals
Sourced in United States
Phenformin is a chemical compound used as a laboratory reagent. It functions as an anti-diabetic agent and is primarily utilized in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Metformin, by Selleck Chemicals (2 mentions)
Rapamycin, by Selleck Chemicals (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Cck8 reagent, by Share-Bio (1 mentions)
Pyrimethamine, by Selleck Chemicals (1 mentions)
Prism 9, by GraphPad (1 mentions)
Lab tek 2 chamber coverglass, by Thermo Fisher Scientific (1 mentions)
Oxaliplatin, by Merck Group (1 mentions)
Carboplatin, by Merck Group (1 mentions)
Azd7545, by Selleck Chemicals (1 mentions)
Azd8055, by Selleck Chemicals (1 mentions)
Etoposide, by Merck Group (1 mentions)
Phenformin hydrochloride, by Merck Group (1 mentions)
Spermidine, by Selleck Chemicals (1 mentions)
Resveratrol, by Selleck Chemicals (1 mentions)
Piperlongumine, by Selleck Chemicals (1 mentions)
Acetylcysteine, by Selleck Chemicals (1 mentions)
Epigallocatechin gallate (egcg), by Selleck Chemicals (1 mentions)
Harmine, by Selleck Chemicals (1 mentions)
Dihydroethidium, by Merck Group (1 mentions)
6 protocols using phenformin
1
SH-SY5Y Cell Culture and Treatment
2
Drug Sensitivity Screening in Cell Lines
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To identify the sensitivities of drugs for different cell lines, the concentration gradient (0, 1, 10, 50, 100, 200, 500, 1000µM) was defined in the Phenformin (Selleck.cn, Shanghai, China) and Pyrimethamine (Selleck.cn, Shanghai, China). The cells were suspended in 100µL DMEM with 10% FBS. 3 × 103 HPDE, MIA PaCa-2 and Patu8988 were seeded into 96-well plates in the different concentration gradient and drugs treatment groups. Then, the 96-well plates were placed into the incubator (5% CO2 at 37 °C) for 24 h. The cells were treated by the Phenformin or Pyrimethamine with different concentration gradient (0, 1, 10, 50, 100, 200, 500, 1000µM) with 36 h. After that, Cell viability was measured by 10% CCK8 reagent (Share-Bio, shanghai, China, SB-CCK8L) at the incubator (5% CO2 at 37 °C) with 1 h. The absorbance of each 96-well plates was measured at 450 and 600 nm by microplate reader, and the half-maximal inhibitory concentration (IC50) was calculated by non-linear regression using GraphPad Prism 9.0.
3
Phenformin Modulates Ovarian Tumor Growth
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Pharmacological grade phenformin was purchased from SelleckChemical, Inc. (Houston, TX, USA). Three weeks after ip ID8 injection, tumor-bearing mice SP+/+ and SP−/− received vehicle (water), or phenformin (1.8 mg/mL) ad lib in drinking water and their daily intake of fluids was monitored. Fresh phenformin was administered every other day. Treatments went on for 3 weeks, through which mice were monitored, as described above, every other day. Mice were euthanized after 2 more weeks, and tissues were harvested and processed as earlier described.
4
Modulating Mitochondrial Dynamics in ESCs
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To inhibit MMP, carbonyl cyanide 3-chlorophenylhydrazone (CCCP, SIGMA ALDRICH, #C2759, 3 µM) was added 24 h after cell plating. All analyses were performed 9 h after CCCP treatment. To inhibit complex I electron transport chain, naive ESCs were treated with phenformin (Selleck Chemical, #S2542, 1 mM) for 2 days. For S-(5′-Adenosyl)-L-methionine chloride dihydrochloride (SIGMA ALDRICH, #A7007, 0.5 mM) supplement, naive ESCs were plated on Lab-Tek II Chamber Coverglass (Thermo Fisher Scientific, #155382). At 24 h post plating, cells were pre-treated with 0.5 mM of SAM or vehicle for 2 h before treatment with CCCP for 6 h. SAM at a final concetration of 25 or 50 µM was used for culturing the spontaneous qESCs for 24 h.
5
Cell Culturing and Drug Treatments
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Cell lines were obtained from the American Tissue Collection Center (ATCC) and grown in high-glucose DMEM (HCT116, U2OS Hela, T47D) or RPMI1640 medium (H460, H1975) supplemented with 10% fetal bovine serum, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin at 37 °C with 5% CO2. All cell lines were authenticated by short tandem repeats profiling and tested negative for mycoplasma contamination. Murine KrasG12D/wt;Trp53Δ/Δ lung adenocarcinoma cells from Adeno-Cre infected KrasLSLG12D/wt;Trp53flox/flox mice33 (link) were cultured in RPMI1640 medium supplemented as above. CDDPnaïve mouse tumor cells were derived from untreated tumors, CDDPres cells from tumors that had relapsed after an initial CDDP treatment. Chemotherapy drugs and inhibitors were obtained from Sigma-Aldrich unless indicated otherwise and used at the following concentrations: CDDP 1 µg ml−1, oxaliplatin 5 µg ml−1, carboplatin 5 µg ml−1, etoposide 1 µm , doxorubicin 0.04 µg ml−1, RITA (Merck) 1 µm , DCA 20–40 mm , 2DG 10–30 mm , metformin 1.25–4 mm , phenformin 60–125 µm , AZD8055 (Selleckchem) 0.2–1 µm , Rapamycin (Selleckchem) 250 nm , Everolimus (Absource Diagnostic) 250 nm , AZD7545 (Selleckchem) 10 µm , chloroquine 25–100 µm , 3-methyladenine (Calbiochem) 5 mm , and SBI-0206965 (Biovision) 5 µm . For starvation experiments, cells were grown in Hank’s Balanced Salt Solution (Sigma) for up to 5 days.
6
Pharmacological Screening of Cellular Modulators
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Acetylcysteine, astaxanthin, epigallocatechin gallate, harmine, LY294002, metformin, moclobemide, phenformin, piperlongumine, rapamycin, resveratrol, sirtinol, spermidine, and tetrahydrocurcumin were purchased from Selleck Chemicals (Houston, TX, USA). rapamycin was purchased from LC Laboratories (Woburn, MA, USA); dihydroethidium, hydroxyurea, and phenformin hydrochloride were obtained from Sigma (St. Louis, MO, USA); and leptomycin B (LMB) was purchased from Alomone Labs (Jerusalem, Israel). AnnH75 was developed and synthesized in the Bracher Lab (Ludwig-Maximilians University, Munich, Germany) (16 (link)).
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