pbabenf2 wild type  (Addgene inc)

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: Tricarbonic acid cycle and glycolysis metabolite level alteration in Nf2 -deficient cells Levels of metabolites as measured by gas chromatography and high performance liquid chromatography. Bolded, underlined numbers indicate significant differences (p ≤ 0.05) between the groups shown, metabolite ratio of <1.00. Greyed, underlined boxes indicate significant difference ( p ≤ 0.05) between the groups shown; metabolite ratio of ≥ 1.00. Two-tailed Welch’s t -test was used to compare FH912 and FC912 cells, n = 5. Two-way ANOVA contrasts were used to compare Nf2 −/− and Nf2 f/f MEFs and to evaluate cerulenin treatment effects.

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

Techniques: Gas Chromatography, High Performance Liquid Chromatography, Phospho-proteomics

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: Upregulation of Fatty Acid Synthesis in Nf2 -deficient cells Samples were processed and analyzed as in .

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

Techniques:

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, Knockdown, Inhibition, Transfection, Electroporation, Negative Control, 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, Phospho-proteomics, Transfection, Plasmid Preparation, Gene Expression, Binding Assay

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 MTOR activation in Nf2−/− vs Nf2f/f MEFs. B) Effect of chemical inhibition of MTOR on cerulenin toxicity. The TORC1 and TORC2 inhibitor Torin1 (25 nM) or TORC1 inhibitor Everolimus (100 nM) were added together with the indicated concentrations of cerulenin, 4 hours after cell seeding, and cells were then incubated for 48 hours. C) Mtor, Rptor and Rictor siRNA transfections. Cells were transfected by electroporation with anti-Mtor, Rptor or Rictor and scrambled (negative control) siRNAs. The indicated amounts of cerulenin were added in 24 hours and incubated for 48 hours. All experiments were repeated 2 times, dose response curves were done in quadruplicates, and for WBs each lysate type was run in duplicates for transfer consistency control. Mean values and 95% confidence intervals are shown on graphs.

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

Techniques: Western Blot, Activation Assay, Inhibition, Incubation, Transfection, Electroporation, Negative Control, Control

Journal: Oncogene

Article Title: KIBRA exhibits MST-independent functional regulation of the Hippo signaling pathway in mammals.

doi: 10.1038/onc.2012.196

Figure Lengend Snippet: Figure 4. KIBRA expression antagonises YAP-induced transcriptional regulation. (a) mRNA expression of the YAP target genes BMP2, FGF1, RASSF4 and PDGFb was assessed by quantitative real-time PCR in MCF10A-YAP cells overexpressing KIBRA, Willin or Merlin. mRNA levels were compared with the empty vector control MCF10A-YAP cells. Willin and KIBRA overexpression increased BMP2 and RASSF4 mRNA levels and decreased FGF1 and PDGFb mRNA levels. Means were calculated from Ct values from two analyses conducted in triplicate. Error bars represent ±s.e. (n ¼ 6). MCF10A-YAP-vector vs MCF10A-YAP-KIBRA or MCF10A-YAP-Willin for all the analysed genes: *Po0.05, **Po0.01, ***Po0.001 Student’s t-test. MCF10A-YAP-vector vs MCF10A-YAP-Merlin: CTGF (P ¼ 0.129), FGF1 (P ¼ 0.005), BMP2 (P ¼ 0.585), RASSF4 (P ¼ 0.307), PDGFb (P ¼ 0.06), Student’s t-test. (b) Immunoblot analysis of KIBRA, Willin or Merlin overexpression in MCF10A-YAP cells, showing 2.7, 1.8 and 8.6-fold increases compared to empty vector control. b-actin was used as a loading control.

Article Snippet: All experiments were carried out at 80–90% confluency. pBABE Nf2 (Johnson et al.45) was obtained from Addgene (plasmid 14116).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Control, Over Expression, Western Blot