Yeast (Saccharomyces cerevisiae) cells were grown in YPD medium (1% yeast extract, 2% peptone and 2% dextrose) at 30 °C with constant shaking at 250 rpm. The wild type parent strain BY4742 (Matα his3Δ leu2Δ met15Δ ura3Δ) and all deletion mutants were purchased from Thermo Scientific (Waltham, MA). For deletion mutants, each gene disruption was replaced with a KanMX module [50 (link)]. For cerulenin treatment, cerulenin (Thermo Scientific, Waltham, MA) stock solution (10 mg/ml in 100% ethanol) was diluted to a final concentration of 4 μg/ml into YPD medium. For controls, the same amount of 100% ethanol was added in place of cerulenin.
Cerulenin
Manufactured by Thermo Fisher Scientific
Cerulenin is a natural product compound that functions as a fatty acid synthase inhibitor. It is commonly used in research applications as a tool compound to study lipid metabolism and biosynthesis pathways.
Automatically generated - may contain errors
Lab products found in correlation
Acridine orange, by Thermo Fisher Scientific (2 mentions)
Total p44 42, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Promokine apoptotic necrotic healthy cell detection kit, by Thermo Fisher Scientific (2 mentions)
Sphingomyelinase fluorometric assay kit, by Cayman Chemical (2 mentions)
Phospho rb ser 608, by Cell Signaling Technology (2 mentions)
Antibodies against glyceraldehyde 3 phosphate dehydrogenase gapdh and hsp70, by Santa Cruz Biotechnology (2 mentions)
Deuterium oxide, by Cambridge Isotopes (1 mentions)
11 bromoundecanoic acid, by Merck Group (1 mentions)
6 protocols using cerulenin
1
Yeast Growth and Cerulenin Treatment
2
Cerulenin-induced Yeast Lipid Synthesis
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Yeast (Saccharomyces cerevisiae) cells were grown in YPD medium (1% yeast extract, 2% peptone and 2% dextrose) at 30°C with constant shaking. The wild type parent strain BY4742 (Matα his3Δ leu2Δ met15Δ ura3Δ) and all deletion mutants were purchased from Thermo Scientific (Waltham, MA). For cerulenin treatment, cerulenin (Thermo Scientific, Waltham, MA) stock solution (10 mg/ml in 100% ethanol) was diluted to a final concentration of 4 µg/ml into YPD medium. For controls, the same amount of 100% ethanol was added in place of cerulenin.
3
Bacillus subtilis 168 Cell Culture
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Cell suspensions of Bacillus subtilis 168 were prepared essentially as previously described (Nickels et al., 2017b (link)). Strain BKE32840 (ΔyusL) was obtained from the Bacillus Genetic Stock Center (The Ohio State University, Columbus, OH, United States). The culture medium used for producing samples was M9 minimal medium containing 0.4% (w/v) glucose and supplemented with 50 mg/L of L-tryptophan (Harwood and Cutting, 1990 ). Solid media were prepared by the addition of 1.5% Noble Agar (Difco). Erythromycin was added to 0.5 μg/mL for routine maintenance of BKE32840. Broth cultures were incubated at 37°C with shaking at 250 rpm. FA feeding experiments included cultures supplemented with 8 mg/L each of a15:0 and n16:0 from 25 mg/mL stock solutions in ethanol, along with 10 g/L of FA-free BSA. Cerulenin (Alfa Aesar) was added to a final concentration of 50 μg/mL. Non-fed cultures were grown in M9 minimal medium with 10 g/L BSA but without FAs or Cerulenin. Cells were harvested at mid-log phase (∼0.8 OD600) by centrifugation at 6,000 × g for 15 min and washed three times in sterile phosphate buffered saline (pH 7.4).
4
Rapamycin and Oleocanthal Synergistic Effects
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Rapamycin was obtained from LC Labs. (-)-OC, extracted from EVOO, was obtained from Dr. Alexios-Leandros Skaltsounis at the University of Athens Department of Pharmacology. The structure and purity (97%) of (-)-oleocanthal was determined by HPLC and 1H-NMR analysis. Acridine orange was obtained from Life Technologies. The Promokine Apoptotic/Necrotic/Healthy cell detection kit and Cerulenin were obtained from Fisher Scientific. The Sphingomyelinase Fluorometric Assay kit was obtained from Cayman Chemicals. Primary antibodies specific for cleaved caspase-3, cleaved PARP, phospho-p44/42 (Thr202/Tyr204), total p44/42, β-actin, and phospho-Rb (Ser 608) were obtained from Cell Signaling Technology. Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Hsp70 were obtained from Santa Cruz Biotechnology.
5
Rapamycin and Oleocanthal Synergistic Effects
6
Synthesis and Characterization of Deuterated Fatty Acids
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Deuterium oxide (99.9% D) and algal amino acids (unlabeled and uniformly D-labeled, with an isotopic purity of 98%) were obtained from Cambridge Isotope Laboratories. Palmitic acid (H-n16:0) and palmitic acid-d31 (D-n16:0) were obtained from Sigma–Aldrich. 12-Methyltetradecanoic acid (H-a15:0) was purchased from Sigma–Aldrich or prepared from s-butyl magnesium chloride (Sigma–Aldrich) and 11-bromoundecanoic acid (Sigma–Aldrich) according to the method of Baer and Carney[64 ] and purified by vacuum distillation. Perdeuterated D-a15:0 was prepared from H-a15:0 through 3 cycles of H/D exchange with D2O catalyzed by 10% Pt/C at 220°C as described by Yepuri et al.,[65 ] followed by chromatography on silica gel and vacuum distillation. The final product was chemically homogeneous and had an isotopic purity of 99%, as determined by analysis of its derived methyl ester by GC/MS. Cerulenin was obtained from Alfa Aesar (Tewskbury, MA) and stored in the dark at –80°C as a solid. FA-free bovine serum albumin (BSA, catalog number A8806) was obtained from Sigma–Aldrich (St. Louis, MO). All other materials were obtained from commercial suppliers and used as received.
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