acknowledgments e14 mouse embryonic stem cells es e14tg2a atcc  (ATCC)

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

Journal: Nature Communications

Article Title: METTL9 sustains vertebrate neural development primarily via non-catalytic functions

doi: 10.1038/s41467-025-62414-5

Figure Lengend Snippet: a Neural differential protocol adopted in this study. Shading indicates the acquisition of mESCs/NSC/NPC identity. b Mettl9 mRNA expression normalised on β-actin (by qPCR), at DIV0, DIV5, DIV10. Error bars are mean ± SD; N = 3. c METTL9 protein expression shown by Western Blot (WB) from Mettl9 WT mESCs DIV0 to DIV10 (anti-METTL9 antibody; kDa: kDalton; N = 1). d Mouse Mettl9 locus and strategy to generate Mettl9 KO mESCs via CRISPR/Cas9 sgRNAs targeting Exon1 (red). e WB from NSC extracts (DIV7) showing endogenous METTL9 expression with an anti-METTL9 antibody (WT; KO: #88 and #90). Blot representative of N = 3, from 3 differentiation experiments. f Representative immunofluorescence (IF) images of Mettl9 WT and Mettl9 KO NSCs (DIV6) with an anti-NESTIN antibody (green) and Hoechst (grey). Scale bar is 10 μm; relative quantification of NESTIN+ cells on the right (t-test, two-sided); error bars are mean ± SD. Quantified cell numbers (n) are shown above each panel (10 fields of view per condition; N = 2 differentiation experiments). g Timepoints of Mettl9 KO mESC neural differentiation (DIV5 and DIV10) analysed by RNA-seq. h Top (10) GO Molecular function terms down-regulated in Mettl9 KO RNA-seq (DIV5). In red, neural-related terms. Hypergeometric test: colour scale shows adjusted p values (Benjamini-Hochberg (BH) correction). i Normalised transcripts per million (TPM) expression of selected basal telencephalic markers. Error bars represent mean ± SE of N = 4. j Top (10) GO Cellular Component terms down-regulated in Mettl9 KO RNA-seq (DIV5; see Methods). Hypergeometric test: colour scale shows adjusted p values (BH correction). k Cell type composition in control WT (E14 or clonal) and Mettl9 KO (#88 and #90) lines inferred by SCADEN deconvolution analysis of scRNA-seq data (DIV10). Asterisk (*) is p = 0.029 (Wilcoxon test; N = 4).

Article Snippet: ES-E14TG2a (E14) mouse embryonic stem cell line (mESC; ATCC), derived from a male 129/Ola mouse blastocyst, were used as wild-type (WT), parental cells.

Techniques: Expressing, Western Blot, CRISPR, Immunofluorescence, Quantitative Proteomics, RNA Sequencing, Control

Journal: Nature Communications

Article Title: METTL9 sustains vertebrate neural development primarily via non-catalytic functions

doi: 10.1038/s41467-025-62414-5

Figure Lengend Snippet: a Experimental strategy for acute METTL9 depletion in Mettl9 Deg mESCs. b Volcano plot of mis-regulated proteins (coloured dots and labels) in dTAG V -1- Mettl9 Deg NSCs over Ctrl (DMSO-treated), by mass spectrometry (q value < 0.05). Y axis indicates log10 FDR-adjusted p values; two-tailed moderated t-statistics. c GO terms of the up-regulated proteins (with a q value < 0.2). Clustering showing significantly enriched GO terms with q value < 0.05. Hypergeometric test; colour scale shows adjusted p values (Benjamini-Hochberg correction). d METTL9 protein: signal peptide (magenta), the glycopeptide position (grey) within sequon (green) and predicted sequon at N86 (aa is amino acid). e WB showing METTL9-DEG in N- (Endo H and PNGase) or O-glycosydase-treated mESCs extracts. Black, green and dark green asterisks (*) are the lowest, intermediate and top METTL9 bands, respectively. Antibodies: anti-FLAG and anti-ACTIN; N = 2 WB and enzymatic treatments. f WB (anti-FLAG, anti-alpha-TUBULIN) showing WT METTL9-FLAG or mutated METTL9-FLAG: N35Q, N35Q;N86Q, and SP* (mutated signal peptide), expressed in WT ESCs. N-glycosylated residues (green); mutated amino acid residues (red); SP (magenta). Asterisks near blot as in ( f ). N = 3 independent experiments. g IF images of Mettl9 Deg NSCs: anti-FLAG (METTL9) and Hoechst (nuclei). Right: a neural rosette. Scale bar: 10 μm. N = 8 fields of view. h IF images of DMSO- (Ctrl) or dTAG V -1-treated Mettl9 Deg NSCs: anti-FLAG antibody showing METTL9. Scale bar: 10 μm. N ≥ 6 fields of view per condition. i IF images of Mettl9 Deg NSCs: anti-FLAG antibody (yellow) for METTL9, GM130 (magenta) for cis -Golgi and Hoechst (cyan) for nuclei. Scale bar: 5 μm. On the right, co-localisation between anti-FLAG and: (i) anti-GM130, (ii) anti-GORASP2 (i and ii for Golgi), (iii) anti-CALRE (ER) or (iv) anti-TOM20 (mitochondria). Boxplot: Manders’ coefficient (M2) indicates co-localization (slices analysed in NO FLAG and FLAG tag, respectively: N = 32, N = 31 (CALRE); N = 35, N = 40, (GM130); N = 35, N = 31 (GORASP2); N = 30, N = 30 (TOM20). P values: above each marker; Wilcoxon test, two-sided. j IF images of Mettl9 Deg /LCS-HA or WT E14 NSCs (DIV5), after Triton or digitonin permeabilization: anti-FLAG antibody (METTL9, yellow), anti-HA (LCS, trans -Golgi lumen, magenta) and Hoechst (nuclei, gray). Scale bar: 10 μm. IFs in ( g – j ) were representative of N ≥ 3 differentiation experiments.

Article Snippet: ES-E14TG2a (E14) mouse embryonic stem cell line (mESC; ATCC), derived from a male 129/Ola mouse blastocyst, were used as wild-type (WT), parental cells.

Techniques: Mass Spectrometry, Two Tailed Test, Glycoproteomics, FLAG-tag, Marker