Journal: Cell Metabolism

Article Title: A Role for p53 in the Adaptation to Glutamine Starvation through the Expression of SLC1A3

doi: 10.1016/j.cmet.2018.07.005

Figure Lengend Snippet: p53 Sustains Aspartate Metabolism under Glutamine Deprivation (A) HCT116 WT and KO clones were cultured in glutamine-free medium for 4 days. Extracellular serine, alanine, and aspartate, normalized to cell number, was quantified over 24 hr. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). (B) Isotope tracing of [ 15 N]aspartate into intracellular glutamate and glutamine. Metabolite percentages in glutamine-deprived HCT116 clones are represented as mean ± SEM of triplicate wells. (C) Isotope tracing of [U- 13 C, 15 N]alanine into intracellular glutamate and glutamine. Metabolite percentages in glutamine-deprived HCT116 clones are presented as mean ± SEM of triplicate wells. (D) Stable isotopomer tracing analysis of [U- 13 C]aspartate incorporation into TCA-cycle intermediates in HCT116 isogenic cells 5 days after glutamine starvation. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). (E) Proliferation of HCT116 WT cells transiently depleted of GOT1 and GOT2 using short interfering RNA (siRNA) and cultured in glutamine-free condition for 6 days. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). The downregulation of these two enzymes was confirmed by western blot analysis (bottom panels). (F) Proliferation of HCT116 WT cells transiently depleted of GOT1 and GOT2 under fully fed conditions. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). (G) Western blot analysis demonstrating the time course of GOT1 and GOT2 expression in HCT116 parental, WT1, and p53-KO1 clones grown in glutamine-free medium over 8 days. .

Article Snippet: Primary antibodies used were as follows: SLC1A3 (5685), AGC1 (64169), MDH2 (11908), phospho-p53 (S15) (9284) from Cell Signaling Technology; p53 (DO-1, sc-126), p21 (sc-397), ACTIN (sc-1616), AGC2 (sc-393303), MDM2 (SMP14, sc-965) from Santa Cruz Biotechnology; GOT1 (ab170950), GOT2 (ab90562), MDH1 (ab180152) from Abcam; glutamine synthetase (610517) from BD Transduction Laboratories.

Techniques: Clone Assay, Cell Culture, Small Interfering RNA, Western Blot, Expressing

Journal: Cell Metabolism

Article Title: A Role for p53 in the Adaptation to Glutamine Starvation through the Expression of SLC1A3

doi: 10.1016/j.cmet.2018.07.005

Figure Lengend Snippet: Deletion of SLC1A3 Impedes the ETC and Phenocopies Depletion of the Mitochondrial Aspartate Transporters AGC1 and AGC2 under Glutamine Deprivation (A) Schematic representation of the malate-aspartate shuttle (MAS). In brief, the MAS is a system that allows the transfer of electrons from cytosolic NADH to produce mitochondrial NADH where it is oxidized in the ETC. In the cytoplasm MDH1 catalyzes the reduction of oxaloacetate (OAA), where it accepts an electron from NADH to produce malate and NAD + . Malate can then enter the mitochondria where it is oxidized by MDH2 to OAA, resulting in the formation of mitochondrial NADH. Mitochondrial OAA is transaminated into aspartate by GOT2 whereby aspartate exits the mitochondria in exchange for cytosolic glutamate through a carrier. OAA is recovered in the cytosol by GOT1. By coupling aspartate-glutamate exchange, the aspartate-glutamate carrier is essential for the shuttle and is thought to represent the rate-limiting step. (B) Respiratory profiles of HCT116 WT cells transiently depleted of SLC1A3 and grown for 24 hr in glutamine-free medium, in the presence of mitochondrial inhibitors (oligomycin, FCCP [carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone] antimycin A/rotenone). Arrows indicate incubation of cells with the indicated inhibitors. Data are presented as mean ± SEM of one representative experiment (n = 6 wells). (C) Oxygen consumption rates (OCR) of HCT116 WT and p53-null clones 2 days after glutamine deprivation as in (B). Data are presented as mean ± SEM of one representative experiment (n = 6 wells). (D) Proliferation of HCT116 WT cells transiently depleted of AGC1 or AGC2 using siRNA and cultured in glutamine-free condition (LHS) or complete medium (RHS). Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). The downregulation of these two proteins was confirmed by western blot (middle panel). (E) HCT116 WT cells transiently depleted of AGC1 or AGC2 using siRNA were grown for 2 days in glutamine-free medium and pulsed with [U- 13 C]aspartate for the final 24 hr. Extracellular levels of aspartate (m+4), alanine, and serine normalized to cell number were quantified over 24 hr. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). (F and G) HCT116 WT cells transiently depleted of AGC1 or AGC2 using siRNA were grown for 2 days in glutamine-free medium and pulsed with [U- 13 C]aspartate for the final 24 hr. Intracellular TCA-cycle intermediates (F) and glutamate and glutamine levels (G) were measured. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). .

Article Snippet: Primary antibodies used were as follows: SLC1A3 (5685), AGC1 (64169), MDH2 (11908), phospho-p53 (S15) (9284) from Cell Signaling Technology; p53 (DO-1, sc-126), p21 (sc-397), ACTIN (sc-1616), AGC2 (sc-393303), MDM2 (SMP14, sc-965) from Santa Cruz Biotechnology; GOT1 (ab170950), GOT2 (ab90562), MDH1 (ab180152) from Abcam; glutamine synthetase (610517) from BD Transduction Laboratories.

Techniques: Incubation, Clone Assay, Cell Culture, Western Blot