Inhibition of acetyl-CoA carboxylase suppresses fatty acid synthesis and tumor growth of non-small c

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established studies that sufficiently accounted for variability among animals to determine a pre-clinical effect. For statistical analysis, variances were similar between all treatment groups. Post hoc analyses from tumor sections. For A549 lung xenograft and GEMM studies, tumor size per treatment was calculated by measuring each individual tumor size from H&E-stained sections using morphometric analysis in Panoramic viewer software (3D Histech). To quantitate lung tumor area, individual tumor sizes were summed for each mouse from each treatment and the percentage of tumor encompassing total lung area was calculated per mouse per treatment. Total lung area was quantitated using Panoramic viewer software. Deuterated water ( 2 H 2 O) in vivo FASyn studies Seven days before euthanization, mice were treated with either vehicle or ND-646 and simultaneously placed onto 2 H 2 O by i.p. injection with 0.035 mL per g of mouse body weight with 0.9% NaCl 2 H 2 O. Drinking water was immediately replaced with 8% 2 H 2 O- enriched water and mice remained on 2 H 2 O for the entire treatment period. Mice were fasted for 6 h before plasma and tissue collection. Plasma enrichment of 2 H 2 O was approximately 5% of body water after 7 days of labeling. Plasma 2 H 2 O enrichment : The 2H labeling of water from samples or standards was determined via deuterium acetone exchange 40,41 . 5 μL of sample or standard was reacted with 4 μL of 10N NaOH and 4 μL of a 5% (v/v) solution of acetone in acetonitrile for 24 hours. Acetone was extracted by the addition of 600 μl chloroform and 0.5 g Na2SO4 followed by vigorous mixing. 100 μL of the chloroform was then transferred to a GCMS vial. Acetone was measured using an agilent DB-35MS column (30 m 3 0.25mm i.d. 3 0.25 mm, Agilent J&W Scientific) installed in an Agilent 7890A gas chromatograph (GC) interfaced with an Agilent 5975C mass spectrometer (MS) with the following temperature program: 60 °C initial, increase by 20 °C/min to 100 °C, increase by 50 °C/min to 220 °C, and hold for 1 min. The split ratio was 40:1 with a helium flow of 1 ml/min. Acetone eluted at approximately 1.5min. The mass spectrometer was operated in the electron impact mode (70 eV). The abundance of mass ions 58–64 was quantified and corrected for natural abundance. Up to six deuterium atoms can transfer to acetone depending on 2 H 2 O enrichment, thus, mole-percent enrichment was quantified and compared to a ten-point standard curve to quantify plasma 2 H 2 O enrichment. Total fatty acids were extracted from tissues and plasma using a bligh and dyer based methanol/chloroform/water extraction with [U- 2 H 31 ] C16:0 (d31-C16:0) as an internal standard. Briefly, 500 μL MeOH, 500 μL CHCL3, 200 μL H 2 O and 10 μL 10 mM d31- C16:0/ 10 mgs tissue were added to weighed pre-ground tissue. This was vortexed for 10 minutes followed by centrifugation at 10,000 g for 5 minutes. The lower chloroform phase was dried and then derivitised to form fatty acid methyl esters (FAMEs) via addition of 500 μL 2% H2SO4 and incubation at 50 °C for 2 hours. FAMEs were extracted via addition of
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  • Winter '19
  • Robert S Kiss
  • Fatty acid metabolism, Nat Med, FASyn

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