Aspartate Aminotransferase Antibody NBP1-54778 from Novus Biologicals, a Bio-Techne Brand

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Nature
April 04 2013

Fig 1: GOT1 is essential for redox balance and growth in PDACa, Relative clonogenic growth of 8988T cells expressing a control shRNA (shGFP) or two independent shRNAs targeting GLUD1, GOT1, GPT2 or PSAT1. Error bars represent s.d. of triplicate wells from a representative experiment. b and h, Relative metabolite abundance in 8988T cells grown in [U-13C5]-Gln upon GOT1 or ME1 knockdown. Data are presented as the total metabolite pool (encompassing both metabolite derived from Gln and that not Gln-derived, left) and the [U-13C]-labeled and Gln-derived metabolite pool (right). Error bars represent the s.d. of three independently prepared samples. c, d, e and f, Relative ROS levels in 8988T cells under conditions indicated as determined by DCFDA staining. DCFDA assay was performed 24hr after supplementing Gln-free media with either OAA (4mM) or dimethyl malate (4mM). Each bar represents the mean of three independent experiments with error bars representing the s.d. g, Schematic depiction of the cytoplasmic reactions that convert Asp into pyruvate. i, NADP+/NADPH ratio in 8988T cells expressing a control shRNA (shGFP), or an shRNA to G6PD, IDH1, GOT1 or ME1. Error bars represent s.d. of five replicate wells from a representative experiment. j, Flux of the Gln carbon skeleton into downstream metabolites as a function of time. The reads for uniformly 13C-labeled metabolites, presented in ion current, are plotted for cells expressing the shGFP control or shRNA to GOT1. Asp, aspartate; OAA, oxaloacetate; Glu, glutamate; GSH, reduced glutathione; GSSH, oxidized glutathione. *, p< 0.05; **, p< 0.01.
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Nature
April 04 2013

Fig 2: Metabolism of the Gln carbon skeleton through GOT1, MDH1 and ME1 supports PDAC growth by maintaining redox balancea and b, Relative clonogenic growth of 8988T cells expressing a control shRNA (shGFP) or two independent shRNAs to MDH1 or ME1. c and e, Relative clonogenic growth of 8988T cells under conditions indicated. Cells were plated in complete culture media (10mM glucose and 2mM Gln), which was replaced the following day with Gln-free medium supplemented with OAA (4mM), GSH (4mM) or N-acetylcysteine (NAC) (4mM). d and f, Relative clonogenic growth of 8988T cells expressing a control shRNA (shGFP) or two independent shRNAs to GOT1 with or without OAA (4mM), GSH (4mM) or NAC (4mM). Error bars represent the s.d. of triplicate wells from a representative experiment. g, Xenograft growth of 8988T cells expressing a control shRNA (shGFP), shRNAs to GOT1 (#1 and #2), MDH1 (#1 and #2) or ME1 (#1 and #2) in mice. Error bars represent s.e.m (n=10). *, p< 0.05; **, p< 0.01.
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Nature
April 04 2013

Fig 3: Oncogenic Kras mediates Gln reprogramming in PDACa, Expression of GLUD1 and GOT1 was determined by quantitative RT-PCR in 8988T cells expressing a control shRNA (shGFP) or two independent shRNAs targeting Kras. Western blot confirmed knockdown of Kras expression. b, The effect of Kras knockdown on GLUD1 or GOT1 protein levels in Panc1 cells expressing a doxycycline-inducible Kras shRNA. c, Relative clonogenic growth of 8988T cells expressing a control shRNA (shGFP) or shRNA to Kras following treatment with AOA or EGCG. Error bars represent the s.d. of triplicate wells from a representative experiment. d, Relative metabolite abundance in Miapaca2 cells grown in [U-13C5]-Gln upon doxycycline-inducible Kras knockdown. Data are presented as the total metabolite pool (encompassing both metabolite derived from Gln and that not Gln-derived, left) and the [U-13C]-labeled and Gln-derived metabolite pool (right). Error bars represent the s.d. of three independently prepared samples. e, Relative cell viability of 8988T cells treated with GLS inhibitors 968 (active) (10µM), 365 (inactive analog) (50µM), or BPTES (100nM) with increasing concentrations of H2O2. . Error bars represent the s.d. of triplicate wells from a representative experiment. f, Model depicting the Kras-regulated Gln metabolic reprogramming in PDAC used to maintain redox and support growth. AOA, aminooxyacetate; EGCG, epigallocatechin gallate. *, p< 0.05; **, p< 0.01.