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

Figure 4. Polyglutamine-tract-containing proteins are depleted from amino-acid-deprived cells and can be recovered by glutaminase inhibition or glutamine addition.

(A) MiaPaCa2 cells were treated as shown for 48 hr. Levels of indicated proteins were examined by western blotting. A representative result (out of three independent experiments) is shown. (B) MiaPaCa2 cells were treated as indicated for 72 hr and nascent RNA synthesis was monitored by 5-ethynyl-uridine (5-EU) incorporation. Levels of indicated polyQ proteins were concurrently measured by western blot. Dotted line represents the relative value of 5-EU incorporation in cells in which transcription was arrested via actinomycin D (ActD) pretreatment for 10 min prior to adding 5-EU. Data are shown as mean ± SD of N = 4 biological replicates. (C) MiaPaCa2 cells were treated with media containing reduced quantities of glutamine or leucine or with complete medium for 48 hr, at which point new protein synthesis was assayed by puromycin incorporation. Levels of indicated polyQ and non-polyQ proteins were determined by western blotting in a parallel set of identically treated samples. A representative result (out of three independent experiments) is shown. (D) MiaPaCa2 cells were transduced with an empty retroviral vector, HA-tagged recombinant TBP (TBP-HA) or HA-tagged recombinant TBP with polyQ tract deleted (ΔpolyQ TBP-HA). Cells were treated as indicated for 48 hr, and levels of recombinant and endogenous TBP were determined by western blot. An asterisk indicates endogenous TBP. A representative result (out of three independent experiments) is shown. (E) A diagram depicting PolyQ-GFP reporter design. (F) Mouse embryonic fibroblasts (MEFs) were transduced with an inducible GFP or PolyQ-GFP construct in a retroviral vector. Cells were treated with complete or amino-acid-free DMEM in presence of doxycycline for 6 hr. GFP fluorescence was measured by FACS. Data are shown as mean ± SD of N = 3 biological replicates. (G) MEFs transduced with the PolyQ-GFP construct in a retroviral vector were treated with doxycycline-containing complete or amino-acid-free DMEM in presence of 1 μM glutaminase inhibitor CB-839, 200 μM L-glutamine + DMSO, or DMSO alone for 6 hr. GFP fluorescence 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 (B,G) or by paired Student’s t test (F). See also Figure 4—figure supplement 1.

Figure 4—source data 1. Summary data and statistics for 5-ethynyl-uridine assays and GFP reporter assays in Figure 4 and Figure 4—figure supplement 1.

Figure 4.

Figure 4—figure supplement 1. Additional data on the recovery of expression of polyglutamine-tract-containing proteins and cellular transcriptional activity via glutaminase inhibition or glutamine supplementation.

Figure 4—figure supplement 1.

(A) MEFs were treated as shown for 48 hr and harvested for western blotting. Protein levels of indicated polyQ proteins were analyzed by western blotting. A representative result (out of N = 5 independent experiments) is shown. (B) Levels of indicated non-polyQ proteins were determined in cells treated as in (A), with CBF1α serving as a polyQ-containing control. A representative result (out of N = 3 independent experiments) is shown. (C) MiaPaCa2 cells were treated as shown for 48 hr and levels of indicated proteins were analyzed by western blotting. A representative result (out of N = 3 independent experiments) is shown. (D, E) MiaPaCa2 cells transduced with shRNAs against GLS or with control shRNAs were treated for 48 hr as shown. Levels of indicated proteins were assessed by western blotting (D), and transcriptional activity (mean ± SD of independent replicates) was measured via 5-EU incorporation assay (E). The data presented are representative of three independent experiments. An asterisk denotes a non-specific band. (F) MiaPaCa2 cells were treated as indicated for 72 hr and relative levels of ribonucleotide triphosphates were determined by mass spectrometry. Data are shown as mean ± SD of N = 4 biological replicates. p-Values (in comparison with 5% AA condition) were calculated by one-way ANOVA with Holm-Sidak post-test. (G) PolyQ-GFP-transduced MEFs were treated with complete or amino-acid-free DMEM for indicated periods of time in the presence of cycloheximide to block new protein production. GFP fluorescence at indicated time points was measured by FACS. Data are shown as mean ± SD of N = 2 biological replicates. p-Values were calculated by one-way ANOVA with Holm-Sidak post-test (E, F).