Journal: Nature Communications

Article Title: Quantitative CRACI reveals transcriptome-wide distribution of RNA dihydrouridine at base resolution

doi: 10.1038/s41467-025-63918-w

Figure Lengend Snippet: a The identified D sites in ct-tRNAs from mouse embryonic stem cells (mESC) using CRACI. b Comparison of modification levels at specific D sites in mESC ct-tRNAs, with violin plots representing the distribution of D stoichiometry at different positions of tRNAs. The D fraction of each site was calculated as the average of two biological replicates. c The identified D sites in mt-tRNAs from mESCs using CRACI. d Quantification of D stoichiometry in mt-tRNAs from mES cells. The identified D sites in Arabidopsis thaliana using CRACI: e ct-tRNAs, f mt-tRNAs, and g chloroplast tRNAs. h Detection of D sites in chloroplast rRNA at position U2467. Venn diagrams illustrating the overlap of D sites in ct-tRNAs across 3 species—human (HepG2), mouse (mESC), and plant ( Arabidopsis thaliana ): i D16/17, j D20, k D47, and l D20a sites. The D fraction of each site was calculated as the average of two biological replicates.

Article Snippet: Human HepG2 cells (HB-8065) and mouse embryonic stem cells (mESCs, CRL-1821) were obtained from the American Type Culture Collection (ATCC).

Techniques: Comparison, Modification

Journal: Cell reports

Article Title: Paraspeckle protein NONO regulates active chromatin by allosterically stimulating NSD1

doi: 10.1016/j.celrep.2025.116247

Figure Lengend Snippet: (A) Schematic illustration of annotated and putative functional domains of NSD1. NID, nuclear receptor interaction domain; PWWP, Pro-Trp-Trp-Pro domain; RBD, RNA-binding domain; PHD, plant homeodomain; AWS, associated with SET domain (also referred to as pre-SET); SET, su(var), enhancer of zeste, trithorax domain. (B) Western blot of NSD1, GAPDH, and H3K36me2 for WT or NSD1/2-dKO HEK293T cells stably rescued with WT or NSD1 mutants. (C) Top: meta-analysis profiling of genome-wide NSD1 and H3K36me2 ChIP-seq signals within −10 kb of TSS to +10 kb of TES. Bottom: representative track images of NSD1-WT or NSD1 ΔPWWP2 in HEK293T NSD1/2-dKO rescued background. TSS, transcription start site; TES, transcription end site. (D) Western blot of NSD1, GAPDH, and H3K36me2 for WT of NSD1/2-dKO HEK293T cells stably rescued with NSD1 or NSD1 PWWP2–4A . (E) Western blot of Nestin, GAPDH, and H3K36me2 for E14-mESC cells undergoing embryoid bodies (EBs) and neural progenitor cell (NPC) differentiation. (F) NPC differentiation of E14-mESCs with indicated genotypes. Top: representative images of (EBs) undergoing NPC differentiation after 3 days of retinoic acid (RA) treatment. Bottom: quantifications of differentiating and non-differentiating EBs. Scale bars, 500 μm.

Article Snippet: Mouse E14Tg2a (E14) Embryonic Stem Cell line , ATCC , Cat# CRL-1821.

Techniques: Functional Assay, RNA Binding Assay, Western Blot, Stable Transfection, Genome Wide, ChIP-sequencing

Journal: Cell reports

Article Title: Paraspeckle protein NONO regulates active chromatin by allosterically stimulating NSD1

doi: 10.1016/j.celrep.2025.116247

Figure Lengend Snippet: (A) Overlay of meta-analysis profiling of H3K36me2 ChIP-seq signals at all genes within a window of −10 kb of TSS to +10 kb of TES in WT and NONO-KO E14-mESC. Representative track images are shown at the bottom. (B) Individual meta-analysis profiling and heatmaps of H3K36me2 ChIP-seq in WT and NONO-KO mESCs. Left: ChIP-seq signals from WT cells were presented at all genes within a −10 kb of TSS to +10 kb of TES window, and NONO-KO cells were aligned to WT cells. Right: ChIP-seq signals were ranked by max peak value and aligned to the centers. (C) qPCR quantification of NEAT1 RNA expression levels in WT and NEAT1 CRISPRi cells. Signals were normalized by GAPDH . n = 5 for each condition. p value was calculated by Student’s t test. Data are presented as mean ± SEM. (D) Immunofluorescence staining of NONO in WT and NEAT1 CRISPRi HEK293T cells. Images were captured under a 63× objective, and the puncta of nuclear paraspeckles were highlighted by red triangles. Scale bars, 50 μm. (E) Quantifications of (D). Nuclear paraspeckles are present in individual WT ( n = 24) and NEAT1 CRISPRi ( n = 40) HEK293T cells. The p value is calculated by chi-squared test. (F) Overlay of meta-analysis profiling of H3K36me2 ChIP-seq signals at all genes within a window of −10 kb of TSS to +10 kb of TES in WT and NEAT1 CRISPRi HEK293T cells. Representative track images are shown at the bottom. (G) Individual meta-analysis profiling and heatmaps of H3K36me2 ChIP-seq in WT and NEAT1 CRISPRi HEK293T cells. ChIP-seq signals from WT cells were presented at all genes within a −10 kb of TSS to +10 kb of TES window, and NONO-KO cells were aligned to WT cells. Right: ChIP-seq signals were ranked by max peak value and aligned to the centers.

Article Snippet: Mouse E14Tg2a (E14) Embryonic Stem Cell line , ATCC , Cat# CRL-1821.

Techniques: ChIP-sequencing, RNA Expression, Immunofluorescence, Staining

Journal: Cell reports

Article Title: Paraspeckle protein NONO regulates active chromatin by allosterically stimulating NSD1

doi: 10.1016/j.celrep.2025.116247

Figure Lengend Snippet: (A) Neural progenitor cell (NPC) differentiation of WT, NSD1-KO, and NONO-KO E14-mESCs. Top: representative images of embryoid bodies (EBs) undergoing NPC differentiation after 3 days of retinoic acid (RA) treatment. Bottom, quantifications of fully differentiated, partially differentiated, or non-differentiated EBs. Scale bars, 500 μm. (B) Heatmaps of differential gene expression analysis in WT, NSD1-KO, and NONO-KO cells treated with RA for 0, 3, or 6 days using RNA-seq. A total of 252 genes associated with neural development and 102 genes associated with stem cell differentiation were presented. (C) Heatmaps of significant changes of gene set enrichment analysis signatures, including stem cell differentiation and neural lineage gene sets in WT compared to NSD1-KO and NONO-KO E14-mESC cells undergoing RA-induced NPC differentiation. (D) Boxplots of log2 fold changes in gene expression using the experimental conditions shown in (B). The box and whisker represent 95%, the third quartile, the median, the first quartile, and 5% distribution of genes. Data are presented as mean ± SEM. p values were calculated by Wilcoxon test ** p < 0.01; *** p < 0.001; and **** p < 0.0001.

Article Snippet: Mouse E14Tg2a (E14) Embryonic Stem Cell line , ATCC , Cat# CRL-1821.

Techniques: Gene Expression, RNA Sequencing, Cell Differentiation, Whisker Assay