Figure 2. Glutaminase inhibition restores charged tRNA^Gln pools in amino-acid-deprived cells.

(A) Depiction of glutamine utilization pathways. (B) A method for a high-throughput profiling of tRNA charging (CHARGE-seq). (C) Mouse embryonic fibroblasts (MEFs) were treated with complete or amino-acid-free DMEM for 6 hr in presence (+) or absence (-) of 1 μM glutaminase inhibitor CB-839 and analyzed by CHARGE-seq. A representative result (out of two independent experiments) is shown. (D) MEFs were treated as in (C) and subjected to tRNA charging assay with qPCR as a readout. Data are shown as mean ± SD of N = 3 biological replicates. p-Values were calculated by one-way ANOVA with Holm-Sidak post-test. (E, F) Western blots of lysates from MEFs treated with complete or amino-acid-free DMEM for 6 hr (E), or with 100% AA (complete DMEM) or 5% AA (each amino acid supplied at a 5% of standard DMEM formulation) DMEM (F) for 24 hr, in presence (+) or absence (-) of 1 μM glutaminase inhibitor CB-839. A representative result (out of three independent experiments) is shown. See also Figure 2—figure supplement 1.

Figure 2—figure supplement 1. Loss of tRNA^Gln charging and its restoration via the inhibition of glutaminase across a variety of cellular contexts.

(A–B) A498 (A) or MiaPaCa2 (B) cells were treated with complete or amino-acid-free DMEM for 6 hr in presence (+) or absence (-) of 1 μM glutaminase inhibitor CB-839. tRNA charging of indicated isodecoders was measured. Data are shown as mean ± SD of N = 2 biological replicates. (C) A498 cells were treated with amino-acid-free DMEM for indicated periods of time in presence (+) or absence (-) of 1 μM glutaminase inhibitor CB-839. Cell lysates were analyzed by western blotting. A representative result (out of three independent experiments) is shown. (D) A498 cells were treated with amino-acid-free DMEM supplied with varied amounts of amino acids as indicated in presence (+) or absence (-) of 1 μM glutaminase inhibitor CB-839 for 24 hr. Cell lysates were analyzed by western blotting. A representative result (out of three independent experiments) is shown. (E, F) MiaPaCa2 cells were treated with complete or amino-acid-free DMEM for 6 hr (E), or with either 100% AA or 5% AA DMEM for 24 hr (F) in the presence (+) or absence (-) of 1 μM glutaminase inhibitor CB-839. Cell lysates were analyzed by western blotting. A representative result (out of three independent experiments) is shown. (G–I) Cell proliferation in 100% AA or 5% AA DMEM in presence (+) or absence (-) of 1 μM glutaminase inhibitor CB-839 was assayed in MEFs (G), A498 (H) and MiaPaCa2 cells (I). Data are shown as mean ± SD of N = 3–4 biological replicates. p-Values were calculated by one-way ANOVA with Holm-Sidak post-test (G–I). (J) MEFs and MiaPaCa2 cells were treated for 48 hr as indicated and cell lysates were analyzed by western blotting. A representative result (out of two independent experiments) is shown.