Fatty acid starvation was achieved by growing the cells in LB until a Klett measurement of 40 (O.D.600~ 0.40). This culture was then used to inoculate a new flask to an initial cell count of 2-5x10 6 x mL -1 and cerulenin (Cayman chemical -Michigan, USA) was added to 10 µg.mL -1 . Assays performed with strains bearing Rel Bs D264G were always performed with 1 mM of IPTG to induce its expression. For the experiments with the P xyl -fabF strains, the pre-inoculum was grown in LB with 0.2% of xylose to the desired density, then 1 mL was taken, washed twice and inoculated in 15 mL of fresh LB without xylose. For the viable cell count, aliquots were taken, serially diluted and plated on LB agar. Plates were then incubated at 37°C overnight for the estimation of CFU/mL. For decoyinine experiments, the drug (Cayman chemical -Michigan, USA) was added at 250 µg.mL -1 together with the addition of cerulenin and viability was estimated 120 minutes later. For FAME complementation assays, cultures were prepared by adding BSA (10 µg.mL -1 , final concentration), cerulenin (10 µg.mL -1 final concentration) and FAME (2.5 µg.mL -1 final concentration) and viability was estimated after 120 minutes. All experiments were performed in triplicate.
Cerulenin
Manufactured by Cayman Chemical
Sourced in United States
Cerulenin is a fatty acid synthase inhibitor commonly used in research applications. It is a natural product isolated from the fungus Acremonium caerulens. Cerulenin inhibits the condensation reaction catalyzed by the β-ketoacyl-ACP synthase (FabB) enzyme, which is a key step in fatty acid biosynthesis.
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Lab products found in correlation
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Spectramax i3 plate, by Molecular Devices (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Mouse ifnγ 485 mi, by R&D Systems (1 mentions)
Siinfekl peptide ova 257 264, by Merck Group (1 mentions)
Monensin solution, by Thermo Fisher Scientific (1 mentions)
Sodium malonate, by Merck Group (1 mentions)
L phenylalanine, by Merck Group (1 mentions)
Lc ms grade, by Merck Group (1 mentions)
L tyrosine, by Thermo Fisher Scientific (1 mentions)
Restriction enzyme, by Thermo Fisher Scientific (1 mentions)
P coumaric acid, by Merck Group (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Synergy 4 hybrid plate reader, by Agilent Technologies (1 mentions)
Platencin, by Cayman Chemical (1 mentions)
Gelcode blue safe protein stain, by Thermo Fisher Scientific (1 mentions)
Dtx 800 multimode detector, by Beckman Coulter (1 mentions)
Prestoblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
Tranylcypromine, by Merck Group (1 mentions)
Homocysteine, by Merck Group (1 mentions)
16 protocols using cerulenin
1
Fatty Acid Starvation and Viability
2
Isolation and culture of primary cortical neurons from Ppt1 model
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For primary cortical neuron cultures, embryos from timed-pregnant, Ppt1-/+ dams were removed, decapitated, and cortices resected at embryonic day (E) 15.5. All dissection steps were performed in ice cold HBSS, pH7.4. Following cortical resection, tissue from each individually-genotyped embryo were digested in HBSS containing 20 U/ml papain and DNAse (20 min total, tubes flicked at 10 min) before sequential trituration with 1 ml (~15 strokes) and 200 μl (~10 strokes) pipettes, generating a single-cell suspension. For live-cell imaging experiments, cells were counted then plated at 150,000–180,000 cells/well in 24-well plates containing poly-D-lysine/laminin-coated coverslips. For biochemical experiments, that is immunoblot, APEGS assay in vitro, cells were plated on poly-D-lysine/laminin-coated 6-well plates at 1,000,000 cells/well. Cells were plated and stored in plating medium (Neurobasal medium containing B27 supplement, L-glutamine and glutamate) for 3–5 DIV, before replacing half medium every 3 days with feeding medium (plating medium without glutamate). Cultures used in chronic palmitoylation inhibitor treatment were exposed to either DMSO (vehicle), 2 BP (1 μm, Sigma, cat: 238422) or cerulenin (1 μm, Cayman Chemicals, cat: 10005647) every 48 hr between DIV 12 and 18.
3
Cerulenin Inhibition of Chiloglottone Production
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All inhibition experiments were conducted on chiloglottone-depleted cut flowers from growth-chamber acclimatized whole plants (C. trapeziformis, C. valida, and C. aff. valida). Stock solutions (40 mM) of Cerulenin (Cayman Chemical, United States) were dissolved in Dimethyl Sulfoximine (Sigma, United States) and diluted to a final concentration of 100 μM with water (assay buffer). An assay buffer without the inhibitor served as the solvent control. The stalks of cut flowers were immersed into either 100 μM of Cerulenin (assay buffer) or solvent control. Next, the tip of each test tube was sealed with parafilm to ensure no direct contact between the solution and the flowers (3 flowers/tube) and then held in a test tube for 3 days. To induce chiloglottone production, UV-B treatments were conducted over a 2-h period on inhibitor-treated and control plants using a custom made light-box following Amarasinghe et al. (2015) (link). The calli of all three species were immediately excised and assayed for chiloglottones as previously described (Falara et al., 2013 (link)). See supplementary materials for detailed chemical analysis methods.
4
Yeast Expression of Drosophila Lipid Genes
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BY4743, a strain mutant for the yeast gene FAA1 (the generous gift of Diane Ward, University of Utah), was transfected with constructs containing Drosophila bgm or dbb cDNAs cloned into the yeast expression vector p426-ADH1. Yeast cultures were grown on YPD (yeast peptone dextrose; standard growth medium) supplemented with cerulenin (Cayman Chemical, Ann Arbor, MI) to inhibit de novo synthesis. Oleate and myristate were obtained from Nu Check Prep (Elysian, MN). Geneticin (G418) selection was employed to monitor the FAA1 mutant background and 5-fluoroorotic acid (5-FOA) was employed to monitor the p426-ADH1 vector.
5
Immunology Reagents for Cell Culture
6
Luciferase-based Tumor-Neutrophil Interaction
7
Analytical Procedures for Chemical Extraction
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All solvents used for solid phase extraction and HPLC-DAD analyses were LC-MS grade from either Sigma-Aldrich (Madrid, Spain) or VWR Chemicals (Barcelona, Spain). Restriction enzymes and T4 DNA ligase were purchased from Thermo Scientific (Madrid, Spain). p-Coumaric acid, L-phenylalanine and sodium malonate were purchased from Sigma-Aldrich (Madrid, Spain), L-tyrosine from Acros Organics (Thermo Fisher, Madrid, Spain), and cerulenin from Cayman Chemicals (Ann Arbor, Michigan, USA).
8
SH-SY5Y Cell Culture and Radiolabeling
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SH-SY5Y cells were a gift from Dr. Colin Combs. All culture media and horse serum were purchased from Life Technologies (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Serum Source International (Charlotte, NC, USA). L-[U 14C] glutamaic acid (260 mCi/mmol) was purchased from PerkinElmer (Waltham, MA, USA). TOFA and cerulenin were purchased from Cayman Chemical (Ann Arbor, MI, USA). All other chemicals and solvents used were purchased from Fisher Scientific (Waltham, MA USA) and were LC-MS grade.
9
Luciferase-based Tumor-Neutrophil Interaction
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Luciferase labeled cells (4T1-Luc, AT3-Luc, MCF7-Luc or MDA-4175-Luc) were seeded in a 24-well plate in 10% FBS/DMEM medium. Cells were allowed to adhere overnight, and were mono-cultured or co-cultured with freshly isolated neutrophils (neutrophils: tumor cells = 20 : 1) in DMEM supplemented with 10% FBS in a direct cell–cell contact manner for 24 hours. The cell culture media was then changed to delipidated media, and the luciferase activity was measured at different time points by using the SpectraMax i3 plate reader (Molecular Devices). For drug treatment experiments, 4T1-Luc cells were seeded and co-cultured with neutrophils as mentioned above. The cell culture medium was then changed to fresh delipidated media containing either DMSO, cerulenin (Cayman chemical) or C75 (Cayman Chemical) at the indicated doses. The final concentration of all wells contained equivalent amounts of DMSO solvent (0.2%). After a 48 hour treatment, the cells were harvested and the luciferase activity was measured by using the SpectraMax i3 plate reader. All values were normalized to the DMSO control sample and performed in triplicate for all cell lines.
10
Biosynthesis of Malonyl-CoA Analogues
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