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: EDC4 , Proteintech , 17737 , 1:100 (immunofluorescence).

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

Journal: iScience

Article Title: Contrastive learning of dynamic processing body formation reveals undefined mechanisms of approved compounds

doi: 10.1016/j.isci.2026.114866

Figure Lengend Snippet: Perturbation of JAK leads to enhanced P-bodies (A) HCT116 cells were knocked down using JAK1 and JAK2 siRNA, and immunostained for P-body components DDX6 (magenta) and EDC4 (green). The nuclei were visualized with DAPI (blue). Scale bar, 10 μm. (B) Quantification of P-body number per cell across three experimental groups. Statistical significance determined by an unpaired t test was indicated as ∗∗∗ p < 0.001. (C) Model of JAK-STAT signaling pathway-mediated P-body regulation. JAK is activated when cytokines or growth factors bind to their respective receptors, leading to receptor dimerization, JAK and STAT phosphorylation, and subsequent transcriptional regulation. Inhibition of the pathway by knockdown of JAK1/2 leads induction of P-body formation. (D) Summary of JAK inhibitors identified in this work that modulate P-body formation.

Article Snippet: As primary antibodies, we used DDX6 rabbit polyclonal antibody (Proteintech, 14632-1-AP) and EDC4 mouse monoclonal antibody (Santa Cruz Biotechnology, sc-376382).

Techniques: Phospho-proteomics, Inhibition, Knockdown

Journal: Nature Communications

Article Title: Systematic characterization of the composition and dynamics of processing body-associated mRNAs

doi: 10.1038/s41467-025-64848-3

Figure Lengend Snippet: a Experimental and analytical workflow of adopting different PB proteins for STAMP and HyperTRIBE. b Immunofluorescence showing subcellular localization of APOBEC1, ADAR2dd, APOBEC1-DDX6, and LSM14A-ADAR2dd proteins with PB marker EDC4 protein, respectively. The experiment was repeated independently for 2 times with similar results. c - c’ Metaplots showing the sequencing depth-normalized distribution of the number of confident edit sites (≥0.65 confidence score) detected in cells expressing PB fusion proteins with high editing efficiency or deaminase alone. c C-to-U edit sites. c’ A-to-I edit sites. d - d’ Volcano plots comparing the editing score (ES) between cells expressing PB fusion protein and those expressing only deaminase. X and Y axes represent log2 fold change (LFC) and −log10( p value), respectively. Red dots represent the target mRNAs passing the respective threshold. Statistical significance was calculated using two-sided Fisher’s exact test followed by Benjamini–Hochberg correction. d APOBEC1-DDX6 vs. APOBEC1. d’ LSM14A-ADAR2dd vs. ADAR2dd. e Venn diagram showing the overlap between target mRNAs identified by APOBEC1-DDX6 and LSM14A-ADAR2dd, respectively. Statistical significance was calculated using the one-sided hypergeometric test. Source data are provided in the Source Data file.

Article Snippet: Antibodies using for the co-IP and western blotting were listed as below: mouse IgG1 isotype antibody (CST, 5415S), MYC tag mouse monoclonal antibody (Proteintech, 66004-1-Ig), HA Tag mouse monoclonal antibody (ABclonal, AE008), EDC4 rabbit polyclonal antibody (Proteintech, 17737-1-AP), HRP-goat anti-mouse IgG(H + L) (Biodragon, BF03001, 1:10,000) and HRP-goat anti-rabbit IgG(H + L) (Biodragon, BF03008, 1:10,000).

Techniques: Immunofluorescence, Marker, Sequencing, Expressing

Journal: Nature Communications

Article Title: Systematic characterization of the composition and dynamics of processing body-associated mRNAs

doi: 10.1038/s41467-025-64848-3

Figure Lengend Snippet: a Immunofluorescence showing localization of APOBEC1, ADAR2dd, and PB marker EDC4 proteins in control HCT116 cells and co-localization of APOBEC1-DDX6 and LSM14A-ADAR2dd proteins with EDC4 proteins in dual-editing HCT116 cells. The experiment was repeated independently for 2 times with similar results. b - b’ Scatter plots showing the correlation of ES between single-editing and dual-editing HCT116 cells. The Pearson correlation coefficient R and p value (two-sided) are indicated in the top left corner. b C-to-U edits. b’ A-to-I edits. c Venn diagram showing the overlap between the target mRNAs identified by APOBEC1-DDX6 and LSM14A-ADAR2dd in dual-editing HCT116 cells. Statistical significance was calculated using the one-sided hypergeometric test. d Venn diagram showing the overlap between the target mRNAs identified by APOBEC1-DDX6 and LSM14A-ADAR2dd in dual-editing HEK293T cells. Statistical significance was calculated using the one-sided hypergeometric test. e Venn diagram showing the overlap between the LSM14A-DDX6-associated mRNAs identified in dual-editing HCT116 and HEK293T cells. Statistical significance was calculated using the one-sided hypergeometric test. f Gene ontology analysis of LSM14A-DDX6-associated mRNAs shared between the two cell lines. Statistical significance was calculated using the clusterProfiler R package with default settings. g Venn diagram showing the overlap between PB-associated mRNAs identified by LSM14A-FAPS, DDX6-eCLIP-seq, and LSM14A-DDX6-associated mRNAs identified by PB-TRIBE-STAMP in HEK293T cells. h - h’ RIP-RT-qPCR validation of LSM14A-DDX6-associated mRNAs in HCT116 cells. Different groups of genes, identified by other methods or exclusively by PB-TRIBE-STAMP are indicated with different colors. Three independent biological replicates were analyzed. h Immunoprecipitation using DDX6 antibody. h’ Immunoprecipitation using EDC4 antibody. Error bars represent mean ± SD. Statistical significance was calculated using ordinary one-way ANOVA (default in Prism 9) (ns: p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001). i - i” RNAscope confocal images showing co-localization of XBP1 mRNAs ( i ), SREBF2 mRNAs ( i’ ), and HMGA2 mRNAs ( I” ) with EDC4 proteins. Source data are provided in the Source Data file.

Article Snippet: Antibodies using for the co-IP and western blotting were listed as below: mouse IgG1 isotype antibody (CST, 5415S), MYC tag mouse monoclonal antibody (Proteintech, 66004-1-Ig), HA Tag mouse monoclonal antibody (ABclonal, AE008), EDC4 rabbit polyclonal antibody (Proteintech, 17737-1-AP), HRP-goat anti-mouse IgG(H + L) (Biodragon, BF03001, 1:10,000) and HRP-goat anti-rabbit IgG(H + L) (Biodragon, BF03008, 1:10,000).

Techniques: Immunofluorescence, Marker, Control, Quantitative RT-PCR, Biomarker Discovery, Immunoprecipitation, RNAscope