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. 2021 Oct 14;10:e71595. doi: 10.7554/eLife.71595

Figure 6. Glutamine and asparagine are utilized for de novo amino acid biosynthesis.

(A–B) Fractional contribution of [U-13C]glutamine or [U-13C]asparagine (A) or [α−15N]glutamine or [α–15N]asparagine (B) to asparagine, aspartate, glutamate, proline, serine, and alanine. (C–D) Fractional contribution of [U-13C]glutamine or [U-13C]asparagine (C) or [α–15N]glutamine or [α–15N]asparagine (D) to asparagine, aspartate, glutamate, proline, serine, and alanine in total protein. (E–F) Effect of BPTES treatment on the fractional contribution of [U-13C]glutamine (E) or [α–15N]glutamine (F) to amino acids. * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.0005, **** p ≤ 0.00005 by ordinary one-way ANOVA with Tukey’s multiple comparisons. (G) Effect of GLS inhibition on intracellular amino acid concentration measured by mass spectrometry. * p ≤ 0.05, multiple unpaired t-tests. Error bars depict SD.(H) Western blot analyses of the effects of BPTES treatment on mTORC1 signaling, Eif2a phosphorylation and COL1A1 expression. Phospho-proteins normalized to respective total protein. COL1A1 normalized to beta-actin. Fold change± SD for three independent experiments. * p ≤ 0.05 by an unpaired two-tailed Student’s t-test. (I) Effect of GLS inhibition on protein synthesis as determined by the rate of 3H Proline incorporation into total protein. * p ≤ 0.05 by an unpaired two-tailed Student’s t-test. Error bars depict SD.

Figure 6—source data 1. Contains numerical and uncropped western blot source data for Figure 6.

Figure 6.

Figure 6—figure supplement 1. Inhibiting glutamine metabolism recapitulates molecular effects of SLC1A5 ablation.

Figure 6—figure supplement 1

(A) Fractional contribution of [U-13C]glutamine or [U-13C]asparagine to fumarate, malate, succinate, and Citrate.
(B) Effect of BPTES treatment on the fractional contribution of [U-13C]glutamine to fumarate, malate, succinate, and Citrate. (C) Effect of BPTES on EdU incorporation. (D) Effect of GLS inhibition on tRNA charging. Error bars depict SD. (E) Gls Crispr targeting strategy. (F) PCR analysis of targeted region. Arrow denotes untargeted Gls band. (G) Western blot analyses of the effect of Gls targeting. sgRNAs targeting luciferase and mCherry (denoted sgLuc) were used as a negative control. * p ≤ 0.05, by an multiple unpaired t-tests. Error bars depict SD.
Figure 6—figure supplement 1—source data 1. Contains numerical and uncropped western blot source data for Figure 6—figure supplement 1.