rabbit anti nono  (Bethyl)

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: The m 6 A modification of circTACC3 regulates its interaction with NONO/p54 nrb . (A) Western blot validation of NONO/p54 nrb pulldown by F2‐circTACC3 in HCCLM3 cells. (B) RIP assay shows enrichment of circTACC3 in NONO/p54 nrb ‐associated RNA in HCCLM3 cells ( n = 4). *** P < 0.001. (C) FLIM‐FRET assay (left panel) and the schematic diagram of FLIM‐FRET (right panel) in PA and OA treated HCCLM3 and HepG2 cells. (D) CLIP assay followed by RT‐PCR gel electrophoresis in HCCLM3 cells. (E) The location of circTACC3 (red), NONO/p54 nrb (green), and LncNEAT1 (magenta) in nuclei (blue) was evaluated in HCCLM3 and HepG2 cells following PA + OA treatment. (F) The distribution of circTACC3 (red) in nuclei (blue) was assessed following NONO/p54 nrb or LncNEAT1 interference in PA and OA treated HCCLM3 and HepG2 cells, respectively. (G) Nucleo‐plasmic fractionation shows the altered intracellular localization of circTACC3 following NONO/p54 nrb and LncNEAT1 interference in PA and OA treated HCCLM3 and HepG2 cells ( n = 4). *** P < 0.001. (H) RIP assay shows the enrichment of circTACC3 in NONO/p54 nrb ‐associated RNA following indicated treatment ( n = 3) in HCCLM3 and HepG2 cells. * P < 0.05; ** P < 0.01; *** P < 0.001; NS , not significant. (I‐K) Representative fluorescence images show the distribution of circTACC3 (red) and NONO/p54 nrb (green) in nuclei (blue) in HCCLM3, HepG2 cells, and Hep3B cells expressed ectopic circTACC3 following indicated treatment. (L) RIP assay shows enrichment of wildtype/m 6 A modification site mutant circTACC3 in NONO/p54 nrb ‐associated RNA in PA and OA treated HCCLM3 and HepG2 cells. Data normalization by dividing anti‐NONO/p54 nrb RIP and IgG RIP values by their respective Input group data ( n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; NS , not significant. Abbreviations: NONO/p54 nrb , non‐POU domain‐containing octamer‐binding protein; CLIP, ultraviolet cross‐linking immunoprecipitation; RT‐PCR, reverse transcription‐PCR; FLIM‐FRET, fluorescence lifetime imaging microscopy‐forster resonance energy transfer; MFD, Minimal Fraction of Donor; PA, palmitic acid; OA, oleic acid; T, tumor tissues; PT, peritumoral normal tissue; NAS, non‐alcoholic fatty liver disease activity score; SAH, S‐adenosylhomocysteine; DAA, 3‐deazaadenosine; Mut, mutation; RIP, RNA immunoprecipitation.

Article Snippet: Then, 10 μg of NONO/p54 nrb Rabbit mAb (#10162S, Cell Signaling Technology, MA, US), or 10 μg of IgG antibody was added to the cell lysate, and the mixture was incubated overnight at 4°C.

Techniques: Modification, Western Blot, Biomarker Discovery, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Fractionation, Fluorescence, Mutagenesis, Binding Assay, Cross-linking Immunoprecipitation, Reverse Transcription, Imaging, Microscopy, Förster Resonance Energy Transfer, Activity Assay, RNA Immunoprecipitation

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: circTACC3‐R loop structure formation in the MASH‐related HCC genome. (A) Schematic representation of procedure to identify circR loops and circTACC3‐R loops. (B) Dot‐blot assay to validate the R loop structure. S9.6 antibody was used to specifically recognize DNA‐RNA hybrids. (C) DRIP assay to identify circTACC3 enrichment in R loop structures in HCCLM3 cells ( n = 4). ** P < 0.01; *** P < 0.001; ND , not detection. (D) Representative fluorescence images show the expression and aggregation of circTACC3 and R loops as glow and spectrum signal intensities in MASLD tissues, MASH‐related HCC tumor tissues and paired peritumoral normal tissues, respectively. (E) The 3D distribution of circTACC3 (red) and R loops (green) were showed by 3D‐FISH‐IF in tissue derived organoids. (F‐I) Representative fluorescence images (F, H) and peak graphs of the linear ROI (region of interest) (G, I) show the location of the indicated molecules in HCC cells following PA + OA treatment. The linear ROI is represented by a solid line in the fluorescence graph. (J) The localization of circTACC3 (red) and S9.6‐stained R loops (green) were validated after NONO/p54 nrb or LncNEAT1 interference in PA and OA treated HCCLM3 and HepG2 cells, respectively. Abbreviations: PA, palmitic acid; OA, oleic acid; DRIP, DNA‐RNA immunoprecipitation; IF, immunofluorescence; FISH, fluorescence i n situ hybridization; MASLD, metabolic dysfunction‐associated steatotic liver disease; MASH, metabolic dysfunction‐associated steatohepatitis; HCC, hepatocellular carcinoma; T, tumor tissues; PT, peritumoral normal tissue; NAS, non‐alcoholic fatty liver disease activity score; NONO/p54 nrb , non‐POU domain‐containing octamer‐binding protein; SAH, S‐adenosylhomocysteine; DAA, 3‐deazaadenosine; Mut, mutation; ROI, region of interest.

Article Snippet: Then, 10 μg of NONO/p54 nrb Rabbit mAb (#10162S, Cell Signaling Technology, MA, US), or 10 μg of IgG antibody was added to the cell lysate, and the mixture was incubated overnight at 4°C.

Techniques: Dot Blot, Fluorescence, Expressing, Derivative Assay, Staining, RNA Immunoprecipitation, Immunofluorescence, Hybridization, Activity Assay, Binding Assay, Mutagenesis

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: circTACC3‐R loop formation is regulated by lipid overload and m 6 A modification. (A) 3D‐distribution of circTACC3 (glow) and S9.6‐stained R loops (spectrum) in nuclei (blue) of MASH‐HCC tissue derived organoids after indicated treatment. (B‐C) DRIP assay shows the enrichment of circTACC3 in R loop structure following m 6 A modification interference in PA and OA treated HCCLM3 and HepG2 cells ( n = 3). * P < 0.05; ** P < 0.01. (D) Representative fluorescence images show S9.6‐stained R loops (green) in PA + OA induced HCC cells transfected with the RNase H1‐Tet‐On system after treatment with or without Dox. (E) Nucleo‐plasmic fractionation shows the altered intracellular localization of circTACC3 in PA and OA treated HCCLM3 and HepG2 cells ( n = 4). *** P < 0.001. (F‐G) Representative fluorescence pictures (F) and peak graphs of the linear ROI (G) demonstrating the colocalization of circTACC3 (red), S9.6‐indicated R loops (green), and NONO/p54 nrb (yellow) in nuclei (blue) with or without Dox‐inducible RNase H1 expression in PA and OA treated HCCLM3 and HepG2 cells. Abbreviations: MASH, metabolic dysfunction‐associated steatohepatitis; HCC, hepatocellular carcinoma; PA, palmitic acid; OA, oleic acid; T, tumor tissue; PT, peritumoral normal tissue; SAH, S‐adenosylhomocysteine; DAA, 3‐deazaadenosine; DRIP, DNA‐RNA immunoprecipitation; Dox, Doxycycline; ROI, region of interest.

Article Snippet: Then, 10 μg of NONO/p54 nrb Rabbit mAb (#10162S, Cell Signaling Technology, MA, US), or 10 μg of IgG antibody was added to the cell lysate, and the mixture was incubated overnight at 4°C.

Techniques: Modification, Staining, Derivative Assay, Fluorescence, Transfection, Fractionation, Expressing, RNA Immunoprecipitation

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: DSB‐circTACC3‐R loops aggregated to promote the inter‐TADs contact. (A) After DRIP‐ChIRP sequencing, the reads distributions across peaks of all independent biological replicates are presented. (B) Genome‐wide distribution of the circTACC3‐R Loop‐located genes positively correlated with PA + OA induction or negatively correlated with m 6 A modification intervention. (C) Representative fluorescence images of the colocalization of indicated molecules in nuclei of HCC cells treated with or without PA and OA following RNase R treatment. (D) Schematic representation shows combination of DRIP‐ChIRP‐seq and γH2AX CUT&Tag‐seq to analyze the distribution of the DSB‐circTACC3‐R Loop structures in the genome. (E) Genome‐wide distribution of the DSB‐circTACC3‐R loop located genes in PA + OA induced HepG2 cells. (F) Top four enriched DSB‐circTACC3‐R loop‐binding motifs based on de novo motif analysis. (G) The dynamic clustering of paraspeckles (indicated by NONO/p54 nrb ‐mCherry) were filmed using STELLARIS Dynamic Signal Enhancement 24 h after PA + OA induction at 5‐min intervals for a duration of 1.5 h. Examples (from 50 min to 85 min) of fusions of several NONO/p54 nrb ‐mCherry foci are shown (time points indicated in minutes). (H) Heatmap depicting the fold change(log 2 ) in Hi‐C contact frequencies between PA + OA‐treated and control cells throughout chromosome 7. Interactions that increase in PA + OA group (red) or decrease in Mock group (blue) are evident. Profile of DRIP‐ChIRP‐seq and γH2AX CUT&Tag‐seq are shown on the top. TADs that had higher inter‐TADs contact frequencies (named “contact‐elevated TADs”) in both long‐range (green box) and between adjacent TADs (red box) are marked. DSB‐circTACC3‐R loops are marked with red arrow. (I) Hi‐C maps around the human STX6 locus that formed DSB‐circTACC3‐R loop structure are shown. DSB‐circTACC3‐R loops are marked with red arrow. Abbreviations: DRIP, DNA‐RNA immunoprecipitation; ChIRP, chromatin isolation by RNA purification; γH2AX, Ser‐139 residue of the histone variant H2AX; CUT&Tag, cleavage under targets and tagmentation; IF, immunofluorescence; FISH, fluorescence i n situ hybridization; PA, palmitic acid; OA, oleic acid; Hi‐C, high‐throughput/resolution chromosome conformation capture; DSB, DNA double‐strand breaks; STX6, Syntaxin 6.

Article Snippet: Then, 10 μg of NONO/p54 nrb Rabbit mAb (#10162S, Cell Signaling Technology, MA, US), or 10 μg of IgG antibody was added to the cell lysate, and the mixture was incubated overnight at 4°C.

Techniques: Sequencing, Genome Wide, Modification, Fluorescence, Binding Assay, Hi-C, Control, RNA Immunoprecipitation, Isolation, Purification, Residue, Variant Assay, Immunofluorescence, Hybridization, High Throughput Screening Assay

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: DSB‐circTACC3‐R loop‐localized genes are selectively activated. (A) List of DSB‐circTACC3‐R loop‐localized genes. (B) DSB‐circTACC3‐loop‐localized genes expression in HepG2 cells with/without lipid overload induction ( n = 4). * P < 0.05; ** P < 0.01; *** P < 0.001; NS , not significant. (C‐D) DRIP‐ChIRP‐seq (C) and γH2AX CUT&Tag‐seq (D) RPKM analysis of DSB‐circTACC3‐loop‐localized genes to compare circTACC3‐R Loop enrichment within the “contact‐elevated TADs” ( n = 26) or not within the “contact‐elevated TADs” ( n = 12). * P < 0.05; ** P < 0.01; *** P < 0.001; NS , not significant. (E‐F) Representative fluorescence images (E) and peaks graphs of the linear ROI (F) show the colocalization of indicated molecules with or without Dox‐inducible RNase H1 expression. (G) NONO/p54 nrb ‐mCherry HepG2 cells with or without Dox‐inducible RNase H1 expression were filmed 24 h after PA + OA induction at 5‐min intervals. Abbreviations: PA, palmitic acid; OA, oleic acid; TAD, topologically associated domain; γH2AX, Ser‐139 residue of the histone variant H2AX; CUT&Tag, cleavage under targets and tagmentation; RPKM, reads per kilobase per million mapped reads; Dox, Doxycycline; ROI, region of interest.

Article Snippet: Then, 10 μg of NONO/p54 nrb Rabbit mAb (#10162S, Cell Signaling Technology, MA, US), or 10 μg of IgG antibody was added to the cell lysate, and the mixture was incubated overnight at 4°C.

Techniques: Expressing, Fluorescence, Residue, Variant Assay

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 experimental flows for proteomics analysis of NSD1-PWWP2’s interactomes. (B) Unique proteins detected by LC-MS and plotted by peptide-spectrum match (PSM) scores against percentage of coverage using DIPG13 (top) and HEK293T (bottom) cells. (C) Illustration of annotated functional domains of NONO. (D) GST pulldown assay of HA-tagged NONO using NSD1-PWWP2 as the bait. Left, pulldown of HA-tagged N-NONO or C-NONO using GST alone or GST-NSD1-PWWP2 followed by western blot of GST and HA. Right, pulldown of HA-tagged N-NONO using GST-NSD1-PWWP2 or GST-NSD1-PWWP2–4A mutant followed by western blot of GST and HA.

Article Snippet: NONO rabbit polyclonal , ProteinTech , Cat# 11058-1-AP.

Techniques: Liquid Chromatography with Mass Spectroscopy, Functional Assay, GST Pulldown Assay, Western Blot, Mutagenesis

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) Demonstration of recombinant protein expression and purification, including NSD1, NSD1 PWWP2–4A , N-NONO, and recombinant di-nucleosomes by Coomassie blue staining. (B) HMT assays of full-length NSD1 or NSD1 PWWP2–4A mutant in an incremental titration of 62.5, 125, and 250 nM. Top: quantifications of autoradiographic signals normalized to NSD1 alone (the second lane). Middle: representative autoradiographic images for stably incorporated [ 3 H]. Bottom: Coomassie blue staining of total nucleosomes. Data are presented as mean ± SEM. (C) HMT assays of 60 nM full-length NSD1 with an incremental titration of N-NONO at 0, 240, 530, 880, and 1760 nM. Top: quantifications of autoradiographic signals normalized to NSD1 alone (the second lane). Middle: representative autoradiographic images for stably incorporated [ 3 H]. Bottom: Coomassie blue staining of total nucleosomes. Data are presented as mean ± SEM. (D) HMT assays of 0.25 μM NSD1 PWWP2–4A mutant with an incremental titration of N-NONO at 0, 530, 880, and 1760 nM. Top: quantifications of autoradiographic signals normalized to NSD1 alone (the second lane). Middle: representative autoradiographic images for stably incorporated [ 3 H]. Bottom, Coomassie blue staining of total nucleosomes. Data are presented as mean ± SEM.

Article Snippet: NONO rabbit polyclonal , ProteinTech , Cat# 11058-1-AP.

Techniques: Recombinant, Expressing, Purification, Staining, Mutagenesis, Titration, Stable Transfection

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: NONO rabbit polyclonal , ProteinTech , Cat# 11058-1-AP.

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) Meta-analysis profiling and heatmaps of NSD1 ChIP-seq signals at all genes within a window of −10 kb of TSS to +10 kb of TES in WT and NONO-KO HEK293T cells. NONO-KO is aligned to WT. (B) Meta-analysis profiling and heatmaps of NONO ChIP-seq signals at all genes within a window of −10 kb of TSS to +10 kb of TES in WT and NSD1-KO HEK293T cells. NSD1-KO is aligned to WT. (C) Meta-analysis profiling and heatmaps of NSD1 ChIP-seq signals at all genes within a window of −10 kb of TSS to +10 kb of TES in WT and NONO-KO mESC cells. NONO-KO is aligned to WT. (D) Meta-analysis profiling and heatmaps of NONO ChIP-seq signals at all genes within a window of −10 kb of TSS to +10 kb of TES in WT and NSD1-KO mESC cells. NSD1-KO is aligned to WT.

Article Snippet: NONO rabbit polyclonal , ProteinTech , Cat# 11058-1-AP.

Techniques: 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) 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: NONO rabbit polyclonal , ProteinTech , Cat# 11058-1-AP.

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