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. 2020 Dec 8;9:e62307. doi: 10.7554/eLife.62307

Figure 5. Amino acid depletion triggers frame shifting within polyglutamine-tract-containing proteins, which can be recovered by glutaminase inhibition or glutamine addition.

(A) A diagram depicting PolyQ−1-GFP reporter design and projected outcomes of an in-frame (relative to a (CAG)n stretch) or a −1 frame shifted translation. (B) Mouse embryonic fibroblasts (MEFs) transduced with PolyQ−1-GFP reporter or empty vector control were treated with complete or amino-acid-free DMEM in presence of 1 μM glutaminase inhibitor CB-839 or DMSO for 9 hr. GFP fluorescence was measured by FACS. A representative result (out of three independent experiments) is shown. (C) MiaPaCa2 cells transduced with PolyQ−1-GFP reporter or empty vector control were treated with 100% AA or 5% AA DMEM in presence or absence of 1 μM glutaminase inhibitor CB-839 for 24 hr. GFP fluorescence was measured by FACS. A representative result (out of at least three independent experiments) is shown. (D) MiaPaCa2 cells transduced with PolyQ−1-GFP reporter were exposed to indicated stressors for 24 hr. GFP fluorescence was measured by FACS. Data are shown as mean ± SD of N = 3 biological replicates. (E) MiaPaCa2 cells transduced with PolyQ−1-GFP or Luc−1-GFP reporter were treated as indicated for 24 hr. GFP accumulation was measured by FACS. Data are shown as mean ± SD of N = 3 biological replicates. (F) PolyQ−1-GFP reporter-transduced MiaPaCa2 cells were treated with 100% AA or 5% AA DMEM for 24 hr, then cultured in 100% AA medium for additional 72 hr. GFP fluorescence was measured by FACS. A representative result (out of three independent experiments) is shown. p-Values were calculated by one-way ANOVA with Holm-Sidak post-test (D, E). See also Figure 5—figure supplement 1.

Figure 5—source data 1. Summary data and statistics for GFP reporter assays presented in Figure 5 and Figure 5—figure supplement 1.

Figure 5.

Figure 5—figure supplement 1. Additional data on the amino acid depletion-triggered translational fidelity loss associated with polyglutamine tracts.

Figure 5—figure supplement 1.

(A, B) PolyQ−1-GFP-transduced MiaPaCa2 (A) or MC38 (B) cells were treated as indicated for 24 hr. GFP accumulation was measured by FACS. A representative result (out of two independent experiments) is shown. (C) PolyQ−1-GFP-transduced MiaPaCa2 cells were treated with DMEM containing indicated levels of amino acids for 24 hr. GFP accumulation was measured by FACS. A representative result (out of two independent experiments) is shown. (D) PolyQ−1-GFP-transduced MiaPaCa2 cells were treated with 100% AA or 5% AA DMEM in presence of 1 μM CB-839, 0.4 μM ISRIB or DMSO as a control for 24 hr. GFP accumulation was measured by FACS. A representative result (out of three independent experiments) is shown. (E) PolyQ−1-GFP- or Luc−1-GFP-expressing MiaPaCa2 cells were treated with indicated media for 48 hr and GFP accumulation was measured by FACS. Data are shown as mean ± SD of N = 3 biological replicates. p-Values were calculated by one-way ANOVA with Holm-Sidak post-test.