Journal: Journal of Cell Science

Article Title: Glutamine modulates stress granule formation in cancer cells through core RNA-binding proteins

doi: 10.1242/jcs.263679

Figure Lengend Snippet: MYC regulates metabolic rewiring during glutamine recovery. (A) RNA FISH in GFP–G3BP1 expressing (magenta) U2OS cells to detect endogenous MYC transcripts (yellow), showing MYC transcripts in SGs, and increased MYC levels after treatments (VBL; EB – EBSS medium). Hoechst 33342 DNA stain is shown in blue. Scale bar: 20 µm. (Right) Endogenous MYC transcripts were quantified (mean±s.e.m., n =3). AA-starved (EB)+VBL treated cells show the highest levels of MYC transcripts, with significant reduction after glutamine addition. Data were analyzed using one-way ANOVA followed by Tukey's post hoc test (** P <0.01, **** P <0.0001). (B) U2OS cells were AA-starved (EB)+VBL, along with glutamine addition and then arsenite and a MYC inhibitor (MYCi), followed by immunofluorescence staining with anti-G3BP1 as a SG marker (magenta) and anti-Dcp1a as a PB marker (green). The presence of the MYC inhibitor prevented all SG formation in AA-starved cells even with glutamine present. Control with DMSO shows cells can form SGs readily without the inhibitor present. Hoechst 33342 DNA stain is shown in blue. Images representative of three repeats. Scale bar: 20 µm. (C) Quantifications of dead cells over time measured by the Cytotox NIR dye under various treatments and arsenite exposure (0.625 µM, sufficient to form SGs), both in U2OS cells with (light curves) or without (dark curves) a MYC inhibitor. Presence of the MYC inhibitor dramatically increased cell death in AA-starved cells, even with glutamine present (mean±s.d., n =3 biological replicates).

Article Snippet: Cell death was quantified using the specialized Incucyte Cytotox NIR Dye (Sartorius, 4846).

Techniques: Expressing, Staining, Immunofluorescence, Marker, Control

Journal: Journal of Cell Science

Article Title: Glutamine modulates stress granule formation in cancer cells through core RNA-binding proteins

doi: 10.1242/jcs.263679

Figure Lengend Snippet: Glutamine recovery and metabolic rewiring occur in a G3BP1/2-dependent manner. (A) Fluorescence images captured in live U2OS cells stably expressing GFP–IGF2BP3. Treatments capable of forming SGs lead to granules formation after ∼1 h, and granule dissolving after ∼2.5 h. Images representative of three repeats. Scale bar: 20 µm. (B) Quantifications of dead cells over time measured by the Cytotox NIR dye under various treatments and arsenite exposure (0.625 µM), both in WT U2OS cells (circles) and ΔΔG3BP1/2 (triangles). WT cells that received glutamine supplementation showed significant increase in cell survival, whereas ΔΔG3BP1/2 cells showed increased cell death in every treatment category (mean±s.d., n =3). (C) NBDG glucose uptake assay in ΔΔG3BP1/2 cells. ΔΔG3BP1/2 cells showed increased glucose uptake compared to WT cells displayed in Fig. 4 . Data were analyzed using one-way ANOVA with Tukey's post hoc test (* P <0.05; ** P <0.01; ns, not significant). (D) smRNA FISH in ΔΔG3BP1/2 U2OS cells to detect endogenous MYC transcripts (yellow), overlayed with Hoechst 33342 DNA stain (blue). Scale bar: 20 µm. (E) Quantification of MYC transcripts using smRNA FISH in ΔΔG3BP1/2 cells (mean±s.d., n =3). KO cells showed decreased MYC expression in arsenite and starved groups compared to WT cells (see Fig. 6A )). (F) Seahorse XF ATP rate assay for quantifying ATP and dividing into glycolytic (red) and mitochondrial (blue) fractions in ΔΔG3BP1/2 cells. KO cells showed little ATP coming from the glycolytic fraction, and even glutamine supplementation, which increases overall ATP, did not change mitochondrial ATP production. Data are mean±s.d., n =3.

Article Snippet: Cell death was quantified using the specialized Incucyte Cytotox NIR Dye (Sartorius, 4846).

Techniques: Fluorescence, Stable Transfection, Expressing, Staining