Primary cells were isolated from vastus lateralis skeletal muscle biopsies derived from healthy volunteers as described (17 (link)). For the high/low glutamine experiment, DMEM glutamine-free medium was used and supplemented to 0.5 or 10 mmol/L final concentration (glutamine; Life Technologies) during the 8-day differentiation period or after differentiation for 48 h. BPTES, a glutaminase 1 inhibitor, was purchased from Sigma-Aldrich (SML0601) and added to the cell medium at a concentration of 10 μmol/L for 24 h. For the silencing experiments, differentiated cells were transfected with 10 nmol/L of either Ambion Silencer Select Negative Control #2 (cat. no. 4390847) or an siRNA against GRB10 (Ambion Silencer Select siRNA s6125; Life Technologies). Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (L2630) and added to the cell medium at a concentration of 100 ng/mL for 24 h.
Sml0601
Manufactured by Merck Group
Sourced in United States
The SML0601 is a laboratory equipment product manufactured by Merck Group. It serves as a benchtop centrifuge, designed for basic separation and concentration applications in research and clinical settings. The core function of the SML0601 is to facilitate the separation of different components within a liquid sample by applying centrifugal force.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dm αkg, by Merck Group (2 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
L2630, by Merck Group (1 mentions)
Cck 8 reagent, by Dojindo Laboratories (1 mentions)
Gw9662, by Cayman Chemical (1 mentions)
S1039, by Selleck Chemicals (1 mentions)
Rapamycin, by Selleck Chemicals (1 mentions)
Lps l3024, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Merck Group (1 mentions)
Etomoxir, by Merck Group (1 mentions)
Uridine, by Merck Group (1 mentions)
E1905, by Merck Group (1 mentions)
Palmitate bsa conjugate, by Merck Group (1 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions)
D4902, by Merck Group (1 mentions)
Uk5099, by Merck Group (1 mentions)
5 protocols using sml0601
1
Skeletal Muscle Cell Isolation and Glutamine Modulation
2
Cell Viability Assay with BPTES and DM-αKG
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Cells were inoculated in 96-well plates with a density of 1000 cells/well. Subsequently, 10 µL of cell counting kit-8 (CCK-8) reagent (Dojindo, Japan) was added in each well followed by culturing in a CO2 incubator for 2 h and absorbance determination. The final concentration of the bis-2-(5-phenylacetamide-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES; SML0601, Sigma, USA) was 10 μmol/L. The final concentration of dimethyl α-ketoglutarate (DM-αKG; 349631, Sigma, USA) was 8 mmol/L.
3
Macrophage Polarization Modulation
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MH-S cells, the SV40 transformed mouse alveolar macrophage cell line (CRI-2019, ATCC, Baltimore, Md, USA), were grown in RPMI-1640 medium (Gibco, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Australia Origin) and 1% penicillin/streptomycin. Cells were cultured at 37°C in 5% CO 2 and 95% airhumidified incubator. MH-S cells were activated as M1 or M2 macrophages with the indicated stimulants (according to previous studies): 1 μg•mL -1 LPS (L3024, Sigma, Mo) 3 h for M1 polarization (16) , and 20 ng•mL -1 of recombinant mouse IL-4 (404-ML, R&D Systems) 8 h for M2 polarization (17) . The Dimethyl 2oxoglutarate (DM-αKG; 349631, Sigma, Mo) (1/2 mM) was added 24 h before LPS or IL-4 stimulation (10) . To further assess the effect of DM-αKG on macrophage polarization, we used Bis-2-(5-phenylacetamido-1,3,4thiadiazol-2-yl) ethyl sulfide (BPTES) (a selective inhibitor of Glutaminase, SML0601, Sigma, Mo)10 μM (10, 18) to inhibit the production of intracellular α-KG. To explore the potential molecular mechanism, we used the GW9662 (a selective PPARγ antagonist, 70785, Cayman) 3 μM (19) and Rapamycin (a prototypical allosteric mTOR inhibitor, S1039, selleckchem, Houston, Tex) 20 nM (20) respectively to inhibit the function of PPARγ and mTORC1. The cell culture and interfering conditions were detailed in Supplementary material (https://links.lww.com/SHK/A853).
4
Investigating BPTES Metabolic Inhibition
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Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) was purchased from Sigma (#SML0601, Sigma-Aldrich (Sigma, St. Louis, MO, USA). The 2mM stock solutions were prepared with DMSO. Then, 10 μM BPTES was added to the cells. DMSO was used as control.
5
Mitochondrial Nutrient Inhibition in Cell Culture
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Cells were cultured under standard conditions of atmospheric O2, 5% CO2 and 37°C in DMEM (Thermo Fisher Scientific #10567022) supplemented with 10% Fetal Bovine Serum (FBS, Thermo Fisher Scientific), 50 μg/mL uridine (Sigma-Aldrich), 1% MEM non-essential amino acids (Thermo Fisher Scientific), and 10 μM-1.7 μM palmitate-BSA conjugate (Sigma-Aldrich). Treatment with dexamethasone (Dex, Sigma-Aldrich #D4902, 100 nM) and Dex plus the Mitochondrial Nutrients Uptake InhibiTors cocktail (Dex+mitoNUITs) began after 15-days of culturing post-thaw to allow the cells to adjust to the in vitro environment and were dosed every passage. The mitoNUITs cocktail included i) UK5099 (Sigma-Aldrich #PZ0160, 2 μM), an inhibitor of the mitochondrial pyruvate carrier (MPC) that interferes with the pyruvate import into the mitochondrial matrix ii) Etomoxir (Sigma-Aldrich #E1905, 4 μM), an inhibitor of carnitine palmitoyltransferase-1 (CPT-1) that interferes with the fattyacid-derived Acyl-CoA import into the mitochondrial matrix; and iii) BPTES (Sigma-Aldrich #SML0601, 3 μM), an inhibitor of glutaminase 1 (GLS1) that prevents the conversion of glutamine to glutamate into the mitochondrial matrix. The combined action of these three compounds ultimately abates the availability of the tricarboxylic acid cycle (TCA) to produce Acetyl-CoA.
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