Single cell suspensions of splenocytes were labeled with CellTrace Violet stain according to the manufacturer’s instructions (Life Technologies and concentrated to 4 × 105–5 × 105 cells in 96 flat-well plates in 200 μl T cell media (RPMI with 10% FCS, 10 mM HEPES, 50 mM 2-mercaptoethanol, 2 mM L-Gln, 1% non-essential amino acids (MEM), 100 units/ml penicillin, 100 mg/ml streptomycin and 1 mM sodium pyruvate) or B-cell media (same as T-cell media but DMEM instead of RPMI). All conditions contained 1 μM 4-OH TAM to induce the HMGCR deletion in vitro. For T-cell stimulation, the conditions contained 1 μg/ml αCD3, and 6 ng/ml αCD28 (BioXCell); for B-cell stimulation 20 μg/ml LPS (Sigma) was added. In some conditions (±)-mevalonolactone (Sigma), a cell-permeable version of GGPP-geranylgeraniol (Sigma), Q-VD-OPh (Sigma) and NBD-6 Cholesterol (Avanti) was used. The cultured cells were analyzed on days 1–3 via FACS analysis.
Mevalonolactone
Manufactured by Merck Group
Sourced in United States, Switzerland, Germany
Mevalonolactone is a chemical compound used as a laboratory reagent. It is a precursor in the biosynthesis of various isoprenoids, such as cholesterol, steroids, and terpenes. Mevalonolactone is commonly used in biochemical and cell biology research applications.
Automatically generated - may contain errors
Lab products found in correlation
Simvastatin, by Merck Group (8 mentions)
Cholesterol, by Merck Group (5 mentions)
Squalene, by Merck Group (5 mentions)
Ym 53601, by Cayman Chemical (4 mentions)
Lovastatin, by Merck Group (4 mentions)
Simvastatin, by Selleck Chemicals (3 mentions)
Mevastatin, by Merck Group (3 mentions)
Paraquat, by Merck Group (3 mentions)
Geranylgeranyl pyrophosphate ammonium salt, by Merck Group (3 mentions)
Pitavastatin, by Selleck Chemicals (3 mentions)
Farnesyl pyrophosphate ammonium salt, by Merck Group (3 mentions)
Pravastatin, by Merck Group (3 mentions)
Fluvastatin, by Selleck Chemicals (2 mentions)
2 hydroxypropyl β cyclodextrin, by Merck Group (2 mentions)
Geranyl pyrophosphate, by Merck Group (2 mentions)
Methionine, by Merck Group (2 mentions)
Uracil, by Merck Group (2 mentions)
Nicotinic acid, by Merck Group (2 mentions)
Glycocholic acid, by Merck Group (2 mentions)
Cortisone, by Merck Group (2 mentions)
47 protocols using mevalonolactone
1
Splenocyte Activation and Analysis
2
Trichodiene Identification and Quantification
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After incubation for 48 h, whole cell cultures of strains TD1-TD4 were extracted with analytical CH2Cl2 for three times, the organic solvents were evaporated to dryness under vacuum, and the residues were dissolved in CH2Cl2 for GC and GC-MS analyses (Dickschat et al., 2011 (link)). Trichodiene was identified using an Agilent 6890N GC coupled with an Agilent 5975 inert XL mass-selective detector (MSD) with a HP-5MS column (25 m × 0.20 mm; 0.33 μm). The oven temperature was set at 60°C for 2 min, increased by 10°C min-1 to 290°C, and held at 290°C for 4 min. The injector temperature was set at 260°C. Helium was used as the carrier gas (0.8 mL min-1) in the splitless mode. Trichodiene was characterized by comparison of its MS data with those published (Dickschat et al., 2011 (link)), and quantified by integrating its peak area in GC chromatogram and comparing to an internal standard, (±)-mevalonolactone (Sigma-Aldrich, St. Louis, MO, United States), which showed similar volatility, but different retention time to trichodiene or other volatiles from yeast.
3
Lipid Metabolism Regulation Assays
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The chemical reagents atorvastatin, lovastatin, fluvastatin, simvastatin, MF-438, diltiazem, carfilzomib, ispinesib, and selinexor were purchased from Selleckchem (Houston, TX, USA). These chemicals were dissolved in DMSO (Calbiochem, San Diego, CA, USA) and diluted to their final concentrations. Mevalonolactone, cholesterol, and oleic acid–albumin were purchased from Sigma-Aldrich; these reagents were dissolved in distilled water.
4
Cell Cycle Synchronization in A549 Cells
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lovastatin was used to synchronize cells in G1 phase. Briefly, culture medium was removed 24 h after the initial plating of A549 cells and replaced with fresh complete medium containing 40 μm lovastatin (Sigma-Aldrich). Cells were incubated in lovastatin for 22 h to achieve cell cycle G0/G1 phase synchronization before other experimental procedures. At time 0, cells were allowed to reenter cell cycle progression by replacing the medium with fresh complete medium supplemented with 4 mm mevalonolactone (Sigma-Aldrich). The synchronization of cells at the G2/M transition by RO-3306 was as follows. At 24 h following plating A549 cells, medium was replaced with fresh complete medium containing 4 μm RO-3306 (Calbiochem) and incubated for 18 h. Cells were then washed three times with PBS and cultured in fresh complete medium to promote cell cycle progression.
5
Comprehensive Glioma Signaling Pathway Analysis
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Smad3 (phos-S208) (Abcam, ab138659), SERPINE1 (Fisher Scientific), Rap 1 (sc-398755) and TGF-β RI Kinase inhibitor V (Santa Cruz Biotechnology), PARP (#9542S), LC3 A/B (#4108S), ZYX, and total Smad3 (Cell Signaling Technology) antibodies were obtained from commercial vendors. Simvastatin was obtained from Sigma Chemical (in vitro stock and in vivo stock for subcutaneous GIC grafts) and Selleckchem (in vivo stock for GIC intracranially grafted mice). TGF-β2, mevastatin, fluvastatin, lovastatin, geranylgeranyl pyrophosphate (GGPP), farnesyl pyrophosphate (FPP) and (±)-mevalonolactone were obtained from Sigma Chemical. Silencer® select pre-designed SMAD3 siRNAs, and Silencer® Select Negative Control siRNA were purchased from Life Technology. LY2109761 was from AdooQ BioScience (A11133).
6
Lipid Modulation in Endothelial Cells
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HCMECs were cultured as described previously before addition of Squalene (#S3626), cholesterol (#C4951), mevalonolactone (#M4667), geranylgeranyl pyrophosphate (GGPP, #G6025), and farnesyl pyrophosphate ammonium salt (FPP, #F6892) (purchased from Sigma, Poole, UK), for 24 hr.
7
Isoprenoid Biosynthesis Precursor Compounds
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All-trans-farnesol, all-trans-geranylgeraniol, farnesyl diphosphate, geranylgeranyl diphosphate, dexamethasone, fosmidomycin, methyl-jasmonate, ethephon and mevalonolactone were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Cellulase RS was obtained from Yakult Pharmaceutical Industry (Tokyo, Japan), and mevinolin was a kind gift from Drs. M. Greenspan and A.W. Alberts (Merck Sharp and Dohme, Rahway, NJ, USA). Before use, the lactone forms of mevinolin and mevalonolactone were converted into their free salt forms, as described by [28 (link)]. 1-deoxy-d -xylulose (DX) was obtained from AlsaChim (Illkirch Graffenstaden, France). (R,S)-[2-14C]mevalonolactone was purchased from Amersham. Analytical-grade solvents were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France), Carlo Erba (Val-de-Reuil, France), or Fischer Scientific France (Illkirch-Graffenstaden).
8
Generating Fluvastatin-Containing Plates
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Fluvastatin-containing plates were made as previously described [7] (link), [8] (link). Briefly, 40 mg fluvastatin was dissolved in 2.31 ml dH2O. Insoluble components were spun down at 5 000×g for 10 min (at 20°C). The supernatant was filter-sterilized and the OD305 nm was measured to determine the final concentration using a standard curve plotted from a known concentration of fluvastatin. Fluvastatin was added directly into NGM media (55°C), to final concentrations of 0.5 mM or 1 mM. Mevalonolactone (Sigma) and paraquat (Sigma) were dissolved in dH2O to produce a 1 M and 100 mM stock solutions, respectively; these were added directly into NGM media (55°C) to achieve the desired concentrations.
9
Breast Cancer Cell Line Culture
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Fluvastatin, caspase-3 inhibitor (Boc-D-FMK) and MG-132 were purchased from Calbiochem. Dulbecco's modified eagles medium (DMEM), Dulbecco's phosphate buffered saline (DPBS), cholera toxin, mevalonolactone, trypan blue, urea, thiourea, CHAPS, DTT, idoacetamide and sodium dodecyl sulphate were purchased from Sigma chemicals and foetal bovine serum (FBS) was from Lonza. IPG linear strips (pH 4–7, 11 cm) and IPG phor buffer were procured from GE healthcare life sciences. BOC-Leu-Arg-Arg-Arg-AMC was purchased from Biomol and all other chemicals used were of reagent grade. All cell lines were purchased from ATCC. MDA-MB-231 and BT-549 (breast adenocarcinoma cell lines) cells were grown in 10% DMEM containing 10% FBS, L-glutamine (4 mmol/L), penicillin (100 units/mL), and streptomycin (100 µg/mL). MCF-10A (non-tumorigenic epithelial cells) were grown in MEBM supplemented with BPE, hEGF, insulin, hydrocortisone and cholera toxin (100 ng/mL). Cells were maintained in an incubator at 37°C under humidified atmosphere with constant supply of 5% CO2.
10
Optimizing Cell Culture Media Components
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Dulbecco’s modified Eagle Medium/Ham’s F-12 (DMEM/F-12, 1:1) was from Hyclone (Thermo-Fisher Scientific, Ottawa, ON). Fetal bovine serum (FBS), normal horse serum (NHS), penicillin/streptomycin, 0.25% trypsin/EDTA, and Hank’s balanced saline solution (HBSS) were from Gibco (Life Technologies, Burlington, ON). Simvastatin was from Calbiochem (EMD Millipore, Billerica, MA). Mevalonolactone, methyl-β-cyclodextrin, and 2-hydroxypropyl-β-cyclodextrin were from Sigma (Oakville, ON). VectaShield mounting medium with 4’, 6-diamidino-2-phenylindole (DAPI) was from Vector Labs (Burlington, ON). All other reagents were of the highest quality available.
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