Journal: Cancer research

Article Title: An essential role for the tumor suppressor Merlin in regulating fatty acid synthesis

doi: 10.1158/0008-5472.CAN-16-2834

Figure Lengend Snippet: A) Cerulenin dose response curve in Nf2−/− and Nf2f/f MEFs, SC4–9 and RT4 cells. Cells were treated with the indicated amounts of cerulenin for 48 hr. Experiments were done in quadruplicates and repeated 4 times. Alamar Blue was used for read-outs. Mean values and 95% confidence intervals are shown on graphs. B) Increased Casp3 cleavage in Nf2−/− MEFs transfected with anti-Fasn siRNA. Cells were transfected by electroporation with anti-Fasn and scrambled (negative control) siRNAs, final concentration 100 nM, and plated into 6-well plates (250,000 cells/well). C) Effect of reintroducing Merlin in Nf2-deficient schwannoma cells. SC4–9 mouse schwannoma cells, transiently transfected with empty pBabe-puro plasmid or pBabe-Merlin plasmid, were tested for sensitivity to cerulenin. All experiments were done in quadruplicates and repeated 4 times. Alamar Blue was used for read-outs. Mean values and 95% confidence intervals are shown on graphs. At 24 h post-transfection, lysates were analyzed by immunoblotting using rabbit anti-FASN and anti-cleaved Casp3 antibodies. Typical blots are shown. Band intensities were quantified using ImageJ software and normalized to GAPDH band intensities. All experiments were repeated 3 times. Mean values and 95% confidence intervals are shown on graphs. **** − p ≤ 0.0001. D–F) Effects of FASN inhibitors GSK2194069, C75, and Luteolin. Experiments were done using the indicated cell types in quadruplicate and repeated 4 times. Alamar Blue was used for read-outs. Mean values and 95% confidence intervals are shown on graphs. G) In vivo effects of cerulenin. 3x106 SC4–9 cells in matrigel:PBS 1:1 per mouse (female nu/nu) were injected subcutaneously. 30 mg/kg/day of cerulenin or 3 mg/kg/day of GSK2194069 in corn oil were given by oral gavage daily starting day 3 after injection; n = 6. Mean values and 95% confidence intervals are shown on graphs.

Article Snippet: Plasmids, antibodies, and reagents pBabe-NF2 was obtained from Addgene.

Techniques: Transfection, Electroporation, Negative Control, Concentration Assay, Plasmid Preparation, Western Blot, Software, In Vivo, Injection

Journal: Cancer research

Article Title: An essential role for the tumor suppressor Merlin in regulating fatty acid synthesis

doi: 10.1158/0008-5472.CAN-16-2834

Figure Lengend Snippet: A) Dose response to FASN inhibitors in primary human schwannoma cells (NF2−/−) compared to normal Schwann cells (NF2+/+). Cells were incubated with indicated concentrations of GSK2194069 for 72 hours. Proliferation (Ki67, first panel), apoptosis (cleaved Casp3, second panel) and merlin status (third panel), were confirmed by immunocytochemistry and confocal microscopy using DAPI for the total cell count and phalloidin for cytoskeleton staining. Quantification of proliferation and apoptosis was performed using ZEN software. B) Dose response in primary human meningioma cells (NF2−/−) to GSK2194069 compared to normal meningeal cells (NF2+/+). Cells were incubated with indicated concentrations of GSK2194069 for 72 hours, proliferation (Ki67, first panel) and apoptosis (cleaved Casp3, second panel) were confirmed by immunocytochemistry using DAPI for the total cell count, merlin status was confirmed by immunoblotting third panel). Experiments were performed in at least triplicates using at least three independent batches of cells from different individuals. # − 0.05 < p < 0.07, * − p < 0.05; ** − p < 0.01. Mean ± s.e.m. is shown on graph.

Article Snippet: Plasmids, antibodies, and reagents pBabe-NF2 was obtained from Addgene.

Techniques: Incubation, Immunocytochemistry, Confocal Microscopy, Cell Counting, Staining, Software, Western Blot

Journal: Cancer research

Article Title: An essential role for the tumor suppressor Merlin in regulating fatty acid synthesis

doi: 10.1158/0008-5472.CAN-16-2834

Figure Lengend Snippet: A) Supplementing culture media with palmitate does not reverse the effect of cerulenin. Palmitate sodium salt (5 μM) was added together with the indicated concentrations of cerulenin, 4 hours after cell seeding, and cells were then incubated for 48 hours. An experiment was done in quadruplicate and repeated 4 times. Mean values and 95% confidence intervals are shown on graphs. B) Cartoon of Palmitate synthetic pathway. Sites of action of small molecule inhibitors are indicated. ACC = acetyl-CoA carboxylase, MCD = malonyl-CoA decarboxylase, ACP = acyl carrier protein, FASN = fatty acid synthase, TOFA = tetradecyloxyfuroic acid. C) Effect of Acaca knockdown on FASN inhibition. Cells were transfected by electroporation with anti-Acaca and scrambled (negative control) siRNAs. The indicated amounts of cerulenin were added in 24 hours and incubated for 48 hours. Parallel transfections for knockdown control were done in a 6-well format. Experiments were done in quadruplicates and repeated 3 times. Mean values and 95% confidence intervals are shown on graphs. D) Effect of Mlycd knockdown on FASN inhibition. Cells were transfected by electroporation with anti-Mlycd and scrambled (negative control) siRNAs. Indicated amounts of cerulenin were added in 24 hours and incubated for 48 hours. Parallel transfections for knockdown control were done in a 6-well format. Experiments were done in quadruplicates and repeated 4 times. Mean values and 95% confidence intervals are shown on graphs. E) Effect of chemical inhibition of ACC on cerulenin toxicity. The ACC inhibitor TOFA (25 μM) was added together with the indicated concentrations of cerulenin, 4 hours after cell seeding, and cells were then incubated for 48 hours. F) Effect of chemical activation of ACC on cerulenin toxicity. The ACC activator 5-iodotubericidin (2.5 μM) was added together with the indicated concentrations of cerulenin, 4 hours after cell seeding, and cells were then incubated for 48 hours. All experiments were done in quadruplicates and repeated 3 times. Mean values and 95% confidence intervals are shown on graphs. G) Effects of Merlin on acyl-CoA levels. UPLC-MS/MS measurements of acetyl-CoA and malonyl-CoA. MEFs were treated with 0.1 μL/mL DMSO or 5 μM cerulenin for 24 hours. Experiments were repeated 3 times. Mean values and 95% confidence intervals are shown on graphs. * − p ≤ 0.05, *** − p ≤ 0.001.

Article Snippet: Plasmids, antibodies, and reagents pBabe-NF2 was obtained from Addgene.

Techniques: Incubation, Inhibition, Transfection, Electroporation, Negative Control, Activation Assay, Tandem Mass Spectroscopy

Journal: Cancer research

Article Title: An essential role for the tumor suppressor Merlin in regulating fatty acid synthesis

doi: 10.1158/0008-5472.CAN-16-2834

Figure Lengend Snippet: A) Immunoblot detection of levels of expression and phosphorylation of lipogenesis-related proteins in Nf2−/− and Nf2f/f MEFs. B) Immunoblot detection of levels of expression and phosphorylation of lipogenesis-related proteins in SC4–9 Babe (SC4–9 cells transiently transfected with empty pBabe-puro plasmid); SC4–9 Merlin (SC4–9 cells transiently transfected with pBabe-Merlin plasmid). Typical blots are shown. C) Lipogenesis gene expression quantification by qPCR. RNA quantification was performed on Nf2−/− and Nf2f/f MEFs; SC4–9 Babe (SC4–9 cells transiently transfected with empty pBabe-puro plasmid); SC4–9 Merlin (SC4–9 cells transiently transfected with pBabe-Merlin plasmid); and Nf2−/− (FH912) and Nf2f/f (FC912) mouse Schwann cells. All experiments were repeated 4 times Mean and 95% CI are shown on graphs. *** − p ≤ 0.001. D) Enzymes involved in fatty acid sythesis. ACC1, Acaca = Acetyl-CoA carboxylase 1; ACC2, Acacb = Acetyl-CoA carboxylase 2; ACL = ATP citrate lyase; SREBP1 = Sterol regulatory element binding protein 1. ACECS1, Acss2 = Acetyl-CoA synthase 1; ACSL1 = Acyl-CoA synthetase long-chain family member 1. Cpt1c = Carnitine palmitoyl transferase Ic. Cpt2 = Carnitine palmitoyl transferase II. Mlycd = Malonyl-CoA decarboxylase.

Article Snippet: Plasmids, antibodies, and reagents pBabe-NF2 was obtained from Addgene.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Binding Assay