Journal: Genes & Development

Article Title: tRNA synthetase activity is required for stress granule and P-body assembly

doi: 10.1101/gad.353535.125

Figure Lengend Snippet: Inhibition of tRNA charging activates the ISR without inducing RNP granules. ( A ) U-2 OS cells were treated with 0.2% DMSO carrier, 20 µM halofuginone (HF), 250 µM sodium arsenite (As), or 1.25 µM thapsigargin (Tg) for 1 h, and Western blotting for phosphorylated and total eIF2α was done. Total protein ( n = 3 independent replicates) and molecular weights (in kilodaltons) are shown. ( B ) Translation activity was measured using bioorthogonal noncanonical amino acid tagging in cells treated as in A . Methionine or azidohomoalanine (AHA) was added 10 min prior to collection. The ratio of nascent to total protein is shown from n = 3 independent replicates. ( C ) U-2 OS cells stably expressing GFP-G3BP1 (green) were treated as in A and fixed, and nuclei were stained with Hoechst (blue). The percentage of cells with SGs was quantified from n = 3 independent experiments; ≥305 cells were counted in each condition. Scale bars, 10 µm. ( D ) Cells were treated as in C , and immunofluorescence and fluorescence in situ hybridization were done to detect UBAP2L (magenta), PABPC1 (cyan), and poly(A) RNA with oligo(dT) probes (yellow) ( n = 2 independent experiments). ( E ) Cells were treated with 0.2% DMSO, 20 µM HF plus 0.2% DMSO, 250 µM arsenite, or 1.25 µM thapsigargin for 1 h, and the percentage of cells with SGs was quantified ( n = 3 independent experiments); ≥287 cells were counted per treatment. ( F ) Cells were treated, and immunofluorescence microscopy was done as in D to detect the P-body marker DCP1A. Quantification of the percentage of cells with P-bodies (PBs) from n = 3 independent experiments is shown; ≥364 cells were counted per treatment. ( G ) Cells were treated as in C to detect XRN1 (magenta) and EDC4 (yellow) by immunofluorescence microscopy ( n = 2 independent experiments). ( H ) U-2 OS cells were treated as in E , and immunofluorescence microscopy was done to detect DCP1A. The percentage of cells with PBs from n = 3 independent experiments is shown; ≥351 cells were counted per treatment across all replicates. Representative images are shown with the average ± SEM for each experiment, and green, gray, and pink points represent the average of each replicate. Statistical significance was assessed with an ordinary one-way ANOVA followed by Tukey's multiple comparisons test. (*) P < 0.05, (**) P < 0.01, (***) P < 0.005, (****) P < 0.001.

Article Snippet: DCP1A , Santa Cruz Biotechnology , SC-100706 , 1:100 (immunofluorescence).

Techniques: Inhibition, Western Blot, Activity Assay, Stable Transfection, Expressing, Staining, Immunofluorescence, Fluorescence, In Situ Hybridization, Microscopy, Marker

Journal: Genes & Development

Article Title: tRNA synthetase activity is required for stress granule and P-body assembly

doi: 10.1101/gad.353535.125

Figure Lengend Snippet: Ribosome release rescues stress-induced RNP granule assembly upon tRNA synthetase inhibition. ( A ) U-2 OS cells stably expressing GFP-G3BP1 were treated with 180 µM emetine (eme) or 0.2% DMSO with 250 µM sodium arsenite (As) for 1 h. Immunofluorescence microscopy was performed for P-body (PB) marker DCP1A, and the SG marker GFP-G3BP1 was imaged. ( Right ) The percentage of cells with SGs or PBs was quantified from n = 3 independent experiments; ≥349 cells were counted per treatment across all replicates. ( B ) Cells were treated with 0.2% DMSO, 20 µM HF, 250 µM As, or 1.25 µM thapsigargin (Tg) with or without 10 µg/mL puromycin for 1 h, followed by imaging and quantification of SGs. The percentage of cells with SGs was quantified from n = 3 independent experiments ( bottom ), and representative images are shown ( top ); ≥350 cells were counted per treatment across all replicates. ( C ) Cells were treated with 20 µM HF in the presence or absence of 250 µM As, 1.25 µM Tg, or 10 µg/mL puromycin for 1 h. The percentage of cells with SGs was quantified from n = 3 independent experiments; ≥290 cells were counted per treatment across all replicates. ( D ) Cells were treated as in B , and IF for DCP1A was performed. The percentage of cells with P-bodies (PBs) was quantified from n = 3 independent experiments ( bottom ), and representative images are shown ( top ); ≥355 cells were counted per treatment across all replicates. ( E ) Cells were treated as in C , and IF was performed to detect DCP1A. The percentage of cells with PBs was quantified from n = 3 independent experiments ( bottom ), and representative images are shown ( top ); ≥339 cells were counted per treatment across all replicates. Quantification is reported as average ± SEM, with green, pink, or gray dots indicating the average of each replicate. Scale bars, 10 µm. Statistical significance was assessed with an ordinary one-way ANOVA followed by Tukey's multiple comparisons test. (*) P < 0.05, (***) P < 0.005, (****) P < 0.001.

Article Snippet: DCP1A , Santa Cruz Biotechnology , SC-100706 , 1:100 (immunofluorescence).

Techniques: Inhibition, Stable Transfection, Expressing, Immunofluorescence, Microscopy, Marker, Imaging

Journal: Genes & Development

Article Title: tRNA synthetase activity is required for stress granule and P-body assembly

doi: 10.1101/gad.353535.125

Figure Lengend Snippet: Amino acid deprivation activates the integrated stress response without inducing stress granules. ( A ) U-2 OS cells were placed in media without arginine (−Arg) for 1, 4, or 16 h or in the same media with arginine added back (+Arg) for 4 h, and a representative Western blot for total and P-eIF2α with total protein is shown. ( B ) As in A , with glutamine (Gln)-free media or with glutamine added back (+Gln). ( C ) U-2 OS cells stably expressing GFP-G3BP1 (green) were treated as in A , followed by immunofluorescence for P-body (PB) marker DCP1A (magenta), and nuclei were stained with Hoechst (blue). The percentages of cells with stress granules (SGs) and PBs were quantified from n = 3 independent experiments; ≥410 cells were counted for each condition. ( D ) As in C but with glutamine deprivation; ≥312 cells were counted for each condition. ( E ) Cells were treated with DMSO or 40 nM thapsigargin (Tg) for 1 h or starved of arginine or glutamine for 4 h followed by Western blotting for total or P-eIF2α, with total protein shown below . ( F ) GFP-G3BP1-expressing U-2 OS cells were treated with DMSO control or 40 nM Tg for 1 h, and immunofluorescence was done for DCP1A. The average percentage of cells with SGs or PBs is shown at the right ; ≥440 cells were counted per treatment. Scale bars, 10 µm. Molecular weights (in kilodaltons) are shown on each blot. Images are shown for each experiment ( n = 3 independent replicates), with quantifications reported as average ± SEM and with green, gray, and pink points representing the value for each replicate. Statistical significance was assessed with an ordinary one-way ANOVA followed by Tukey's multiple comparisons test for all but F , where a t -test was used. (*) P < 0.05, (**) P < 0.01, (****) P < 0.001.

Article Snippet: DCP1A , Santa Cruz Biotechnology , SC-100706 , 1:100 (immunofluorescence).

Techniques: Western Blot, Stable Transfection, Expressing, Immunofluorescence, Marker, Staining, Control

Journal: microPublication Biology

Article Title: RanGAP1 accumulates in stress-induced cytoplasmic compartments that are distinct from stress granules and P-bodies

doi: 10.17912/micropub.biology.002003

Figure Lengend Snippet: (A-C) Cells were treated with the vehicle DMSO or PFT-µ for 2 h. Samples were then processed for immunostaining or Western blotting. (A-B) Images were acquired by conventional fluorescence or super-resolution microscopy, as indicated. Size bars are 20 µm. (A) RanGAP1 was detected by immunostaining; G3BP1 was used as SG marker. Granule parameters were quantified for three independent experiments. The granule area in µm 2 was measured for 20 granules per experiment. The number of granules per cell [No] was determined for 20 cells for each experiment. Bar graphs depict the averages ± standard error of the mean. Student’s t-test revealed significant differences for the number of granules/cell; *, p < 0.05. Scatter plots display results for individual granules or cells. (B) RanGAP1 biomolecular condensates were compared with P-bodies, using the marker protein Dcp1. (C) Western blotting of crude cell extracts evaluated the SUMOylation and abundance of RanGAP1. A representative blot of four independent experiments is shown. The molecular mass of marker proteins is depicted at the left margin. The ratio non-SUMOylated/SUMOylated RanGAP1 was normalized to vehicle controls (light gray bars). The abundance of total RanGAP1 (SUMOylated RanGAP1 + non-SUMOylated RanGAP1) relative to actin is displayed as dark gray bars. Data are shown for four independent experiments as averages ± standard error of the mean. Significant differences were identified with Student’s t-test; *, p < 0.05; **, p < 0.01. Veh, vehicle; PFT-µ, pifithrin-µ; AU, arbitrary units.

Article Snippet: Dcp1 , Santa Cruz Biotechnology , sc-100706 , NA , 1:200.

Techniques: Immunostaining, Western Blot, Fluorescence, Super-Resolution Microscopy, Marker