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Genechem flag tagged nono
Flag Tagged Nono, supplied by Genechem, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/flag+tagged+nono/pmc13137859-215-0-11?v=Genechem
Average 86 stars, based on 1 article reviews

flag tagged nono - by Bioz Stars, 2026-06

86/100 stars

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(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.

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: 1–217, and a.a. 218–466, of human NONO were amplified from NONO cDNA (SinoBiological, catalog no. HG14826-CF) and cloned to pFASTBac1 with 1× FLAG tag at the N terminus end.

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

(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.

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: 1–217, and a.a. 218–466, of human NONO were amplified from NONO cDNA (SinoBiological, catalog no. HG14826-CF) and cloned to pFASTBac1 with 1× FLAG tag at the N terminus end.

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

(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.

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: 1–217, and a.a. 218–466, of human NONO were amplified from NONO cDNA (SinoBiological, catalog no. HG14826-CF) and cloned to pFASTBac1 with 1× FLAG tag at the N terminus end.

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

(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.

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: 1–217, and a.a. 218–466, of human NONO were amplified from NONO cDNA (SinoBiological, catalog no. HG14826-CF) and cloned to pFASTBac1 with 1× FLAG tag at the N terminus end.

Techniques: ChIP-sequencing

(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.

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: 1–217, and a.a. 218–466, of human NONO were amplified from NONO cDNA (SinoBiological, catalog no. HG14826-CF) and cloned to pFASTBac1 with 1× FLAG tag at the N terminus end.

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

Circ-hnRNPU interacts with and induces cytoplasmic retention of NONO in gastric cancer cells. a , Coomassie bright blue staining (left panel) and Venn diagram (right panel) showing the differential proteins pulled down by biotin-labeled sense (S) or antisense (AS) probe targeting junction sites of circ-hnRNPU from the lysates of AGS cells, and overlapping analysis of proteins identified by mass spectrometry (MS) with established RBP from RBPDB ( http://rbpdb.ccbr.utoronto.ca ) and c-Myc-binding proteins derived from BioGRID and IntAct ( https://www.ebi.ac.uk/intact ) databases. b , Western blot (upper panel) and RT-PCR (lower panel) assays indicating the NONO protein or circ-hnRNPU pulled down by biotin-labeled S or AS probes targeting junction site of endogenous circ-hnRNPU from lysates of AGS cells. c , RIP (upper panel) and western blot (lower panel) assays using NONO antibody showing the interaction of NONO with circ-hnRNPU or hnRNPU mRNA in MKN-45 and AGS cells stably transfected with empty vector (mock) or circ-hnRNPU . d , RNA EMSA determining the interaction between recombinant GST-tagged NONO protein and biotin-labeled circular probe of circ-hnRNPU , with or without competition using an excess of unlabeled circular probe. e , Schematic diagram of NONO truncations (left panel) and in vitro binding assay (middle and right panels) depicting the recovered circ-hnRNPU levels detected by RT-PCR after incubation with full-length or truncation forms of GST-tagged or Flag-tagged recombinant NONO protein validated by western blot. f – h , Representative images and quantification of dual RNA-FISH and immunofluorescence ( f ), real-time qRT-PCR ( g , normalized to β-actin ), and western blot ( h ) assays indicated the localization of circ-hnRNPU and NONO (arrowheads), transcript and protein levels of NONO in MKN-45 and AGS cells stably transfected with mock, circ-hnRNPU , lin-hnRNPU , scramble shRNA (sh-Scb), or sh- circ-hnRNPU . Scale bar: 10 μm. Student’s t test compared the difference in f and g . ** P < 0.01. Data are representative of three independent experiments in b - h

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circ-hnRNPU inhibits NONO-mediated c-Myc transactivation and mRNA stabilization essential for glycosylation and cancer progression

doi: 10.1186/s13046-023-02898-5

Figure Lengend Snippet: Circ-hnRNPU interacts with and induces cytoplasmic retention of NONO in gastric cancer cells. a , Coomassie bright blue staining (left panel) and Venn diagram (right panel) showing the differential proteins pulled down by biotin-labeled sense (S) or antisense (AS) probe targeting junction sites of circ-hnRNPU from the lysates of AGS cells, and overlapping analysis of proteins identified by mass spectrometry (MS) with established RBP from RBPDB ( http://rbpdb.ccbr.utoronto.ca ) and c-Myc-binding proteins derived from BioGRID and IntAct ( https://www.ebi.ac.uk/intact ) databases. b , Western blot (upper panel) and RT-PCR (lower panel) assays indicating the NONO protein or circ-hnRNPU pulled down by biotin-labeled S or AS probes targeting junction site of endogenous circ-hnRNPU from lysates of AGS cells. c , RIP (upper panel) and western blot (lower panel) assays using NONO antibody showing the interaction of NONO with circ-hnRNPU or hnRNPU mRNA in MKN-45 and AGS cells stably transfected with empty vector (mock) or circ-hnRNPU . d , RNA EMSA determining the interaction between recombinant GST-tagged NONO protein and biotin-labeled circular probe of circ-hnRNPU , with or without competition using an excess of unlabeled circular probe. e , Schematic diagram of NONO truncations (left panel) and in vitro binding assay (middle and right panels) depicting the recovered circ-hnRNPU levels detected by RT-PCR after incubation with full-length or truncation forms of GST-tagged or Flag-tagged recombinant NONO protein validated by western blot. f – h , Representative images and quantification of dual RNA-FISH and immunofluorescence ( f ), real-time qRT-PCR ( g , normalized to β-actin ), and western blot ( h ) assays indicated the localization of circ-hnRNPU and NONO (arrowheads), transcript and protein levels of NONO in MKN-45 and AGS cells stably transfected with mock, circ-hnRNPU , lin-hnRNPU , scramble shRNA (sh-Scb), or sh- circ-hnRNPU . Scale bar: 10 μm. Student’s t test compared the difference in f and g . ** P < 0.01. Data are representative of three independent experiments in b - h

Article Snippet: The GST- or Flag-tagged NONO protein and biotin-labeled circular probe of circ-hnRNPU were pulled down by using Flag or GST beads (Sigma).

Techniques: Staining, Labeling, Mass Spectrometry, Binding Assay, Derivative Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Transfection, Plasmid Preparation, Recombinant, In Vitro, Incubation, Immunofluorescence, Quantitative RT-PCR, shRNA

Circ-hnRNPU inhibits interaction of NONO with c-Myc in gastric cancer cells. a, Immunofluorescence assay showing the localization of NONO and c-Myc protein (arrowheads) in AGS cells stably transfected with empty vector (mock), NONO , or c-Myc . Scale bar: 10 μm. b , Schematic diagram, co-IP and western blot assays indicating the interaction between full-length or truncations of recombinant GST-tagged c-Myc and MBP-tagged NONO proteins. c , Co-IP and western blot assays revealing the direct interaction between recombinant MBP-tagged NONO and GST-tagged c-Myc proteins, with or without treatment by in vitro generated circ-hnRNPU . d , BiFC assay showing the interaction between NONO and c-Myc (arrowheads) in AGS cells stably transfected with mock, circ-hnRNPU , scramble shRNA (sh-Scb), or sh-circ-hnRNPU #1. Scale bar: 10 μm. e , Representative images (left panel) and quantification (right panel) of immunofluorescence assay revealing the co-localization of NONO and c-Myc (arrowheads) in AGS cells stably transfected with mock or circ-hnRNPU . Scale bar: 10 μm. f , Immunofluorescence (lower left panel), RIP and western blot (lower right panel) assays showing the localization (arrowheads) and interaction of NONO with circ-hnRNPU or c-Myc protein in AGS cells transfected with Flag-tagged wild-type (WT) or NLS-mutant (mNLS) form of NONO as indicated (upper panel). Student’s t test compared the difference in e . ** P < 0.01. Data are representative of three independent experiments in a - f

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circ-hnRNPU inhibits NONO-mediated c-Myc transactivation and mRNA stabilization essential for glycosylation and cancer progression

doi: 10.1186/s13046-023-02898-5

Figure Lengend Snippet: Circ-hnRNPU inhibits interaction of NONO with c-Myc in gastric cancer cells. a, Immunofluorescence assay showing the localization of NONO and c-Myc protein (arrowheads) in AGS cells stably transfected with empty vector (mock), NONO , or c-Myc . Scale bar: 10 μm. b , Schematic diagram, co-IP and western blot assays indicating the interaction between full-length or truncations of recombinant GST-tagged c-Myc and MBP-tagged NONO proteins. c , Co-IP and western blot assays revealing the direct interaction between recombinant MBP-tagged NONO and GST-tagged c-Myc proteins, with or without treatment by in vitro generated circ-hnRNPU . d , BiFC assay showing the interaction between NONO and c-Myc (arrowheads) in AGS cells stably transfected with mock, circ-hnRNPU , scramble shRNA (sh-Scb), or sh-circ-hnRNPU #1. Scale bar: 10 μm. e , Representative images (left panel) and quantification (right panel) of immunofluorescence assay revealing the co-localization of NONO and c-Myc (arrowheads) in AGS cells stably transfected with mock or circ-hnRNPU . Scale bar: 10 μm. f , Immunofluorescence (lower left panel), RIP and western blot (lower right panel) assays showing the localization (arrowheads) and interaction of NONO with circ-hnRNPU or c-Myc protein in AGS cells transfected with Flag-tagged wild-type (WT) or NLS-mutant (mNLS) form of NONO as indicated (upper panel). Student’s t test compared the difference in e . ** P < 0.01. Data are representative of three independent experiments in a - f

Article Snippet: The GST- or Flag-tagged NONO protein and biotin-labeled circular probe of circ-hnRNPU were pulled down by using Flag or GST beads (Sigma).

Techniques: Immunofluorescence, Stable Transfection, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Western Blot, Recombinant, In Vitro, Generated, Bimolecular Fluorescence Complementation Assay, shRNA, Mutagenesis

Circ-hnRNPU inhibits dual NONO activity in regulating c-Myc transactivation and mRNA stabilization. a and b , Dual-luciferase assay using a reporter containing three canonical c-Myc binding sites ( a ), ChIP and qPCR (b, normalized to input) assays indicating c-Myc transactivation and enrichment on target gene promoters in MKN-45, AGS, and HeLa cells stably transfected with empty vector (mock) or circ-hnRNPU , and those co-transfected with NONO or c-Myc ( n = 5). c and d , Dual-luciferase assay ( c ), western blot (d, left panel), and real-time qRT-PCR (d, normalized to β-actin , right panel) assays showing promoter activity or expression of GALNT6 and MGAT1 , as well as circ-hnRNPU or downstream targets ( FN1 and GLUT1 ) levels in AGS cells stably transfected with mock or circ-hnRNPU , and those co-transfected with NONO or c-Myc . e , Venn diagram (left pane) and CLIP-seq peak (middle panel) indicating comprehensive analysis of 112 altered genes in RNA-seq and NONO-binding targets (GSE90650). RIP and real-time qRT-PCR assays (right panel) revealing endogenous NONO binding to 3'-UTR of hnRNPU , GALNT2 , and GALNT6 in AGS cells. f and g , Dual-luciferase assay using a 3'-UTR reporter containing four canonical NONO binding sites (f), RIP and real-time qRT-PCR (g) assays showing the activity and enrichment of NONO on 3'-UTR of hnRNPU , GALNT2 , and GALNT6 in MKN-45, AGS, and HeLa cells stably transfected with mock or circ-hnRNPU , and those co-transfected with NLS-mutant (mNLS) form of NONO ( n = 5) . h , Real-time qRT-PCR (normalized to β-actin , n = 5) assays showing the mRNA stability of hnRNPU , GALNT2 , and GALNT6 in AGS cells stably transfected with mock or circ-hnRNPU , and those co-transfected with mNLS form of NONO or treated with actinomycin D (5 μg/ml) as indicated. i , Western blot assay indicating the expression of hnRNPU, GALNT2, and GALNT6 in AGS cells stably transfected with mock or circ-hnRNPU , and those co-transfected with mNLS form of NONO. Student’s t test or analysis of variance compared the difference in a - h . * P < 0.05, ** P < 0.01. Data are shown as mean ± s.e.m. (error bars) or representative of three independent experiments in a - i

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circ-hnRNPU inhibits NONO-mediated c-Myc transactivation and mRNA stabilization essential for glycosylation and cancer progression

doi: 10.1186/s13046-023-02898-5

Figure Lengend Snippet: Circ-hnRNPU inhibits dual NONO activity in regulating c-Myc transactivation and mRNA stabilization. a and b , Dual-luciferase assay using a reporter containing three canonical c-Myc binding sites ( a ), ChIP and qPCR (b, normalized to input) assays indicating c-Myc transactivation and enrichment on target gene promoters in MKN-45, AGS, and HeLa cells stably transfected with empty vector (mock) or circ-hnRNPU , and those co-transfected with NONO or c-Myc ( n = 5). c and d , Dual-luciferase assay ( c ), western blot (d, left panel), and real-time qRT-PCR (d, normalized to β-actin , right panel) assays showing promoter activity or expression of GALNT6 and MGAT1 , as well as circ-hnRNPU or downstream targets ( FN1 and GLUT1 ) levels in AGS cells stably transfected with mock or circ-hnRNPU , and those co-transfected with NONO or c-Myc . e , Venn diagram (left pane) and CLIP-seq peak (middle panel) indicating comprehensive analysis of 112 altered genes in RNA-seq and NONO-binding targets (GSE90650). RIP and real-time qRT-PCR assays (right panel) revealing endogenous NONO binding to 3'-UTR of hnRNPU , GALNT2 , and GALNT6 in AGS cells. f and g , Dual-luciferase assay using a 3'-UTR reporter containing four canonical NONO binding sites (f), RIP and real-time qRT-PCR (g) assays showing the activity and enrichment of NONO on 3'-UTR of hnRNPU , GALNT2 , and GALNT6 in MKN-45, AGS, and HeLa cells stably transfected with mock or circ-hnRNPU , and those co-transfected with NLS-mutant (mNLS) form of NONO ( n = 5) . h , Real-time qRT-PCR (normalized to β-actin , n = 5) assays showing the mRNA stability of hnRNPU , GALNT2 , and GALNT6 in AGS cells stably transfected with mock or circ-hnRNPU , and those co-transfected with mNLS form of NONO or treated with actinomycin D (5 μg/ml) as indicated. i , Western blot assay indicating the expression of hnRNPU, GALNT2, and GALNT6 in AGS cells stably transfected with mock or circ-hnRNPU , and those co-transfected with mNLS form of NONO. Student’s t test or analysis of variance compared the difference in a - h . * P < 0.05, ** P < 0.01. Data are shown as mean ± s.e.m. (error bars) or representative of three independent experiments in a - i

Article Snippet: The GST- or Flag-tagged NONO protein and biotin-labeled circular probe of circ-hnRNPU were pulled down by using Flag or GST beads (Sigma).

Techniques: Activity Assay, Luciferase, Binding Assay, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Expressing, RNA Sequencing Assay, Mutagenesis

Circ-hnRNPU inhibits protein glycosylation, growth, and invasion of gastric cancer cells by repressing NONO or c-Myc activity. a and b , Western blot assay showing the levels of O- and N-glycosylation in MKN-45 and AGS cells stably transfected with empty vector (mock), circ-hnRNPU , scramble shRNA (sh-Scb), or sh- circ-hnRNPU #1, and those co-transfected with NONO , c-Myc , sh-NONO #1, or sh-c-Myc #1. c and d , Representative images (left) and quantification (right) of soft agar ( c ) and matrigel invasion ( d ) assays indicating the anchorage-independent growth and invasion of MKN-45 and AGS cells stably transfected with mock, circ-hnRNPU , sh-Scb, or sh- circ-hnRNPU #1, and those co-transfected with NONO , c-Myc , sh-NONO #1, or sh-c-Myc #1. Student’s t test compared the difference in c and d . * P < 0.05. Data are shown as mean ± s.e.m. (error bars) or representative of three independent experiments in a - d

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circ-hnRNPU inhibits NONO-mediated c-Myc transactivation and mRNA stabilization essential for glycosylation and cancer progression

doi: 10.1186/s13046-023-02898-5

Figure Lengend Snippet: Circ-hnRNPU inhibits protein glycosylation, growth, and invasion of gastric cancer cells by repressing NONO or c-Myc activity. a and b , Western blot assay showing the levels of O- and N-glycosylation in MKN-45 and AGS cells stably transfected with empty vector (mock), circ-hnRNPU , scramble shRNA (sh-Scb), or sh- circ-hnRNPU #1, and those co-transfected with NONO , c-Myc , sh-NONO #1, or sh-c-Myc #1. c and d , Representative images (left) and quantification (right) of soft agar ( c ) and matrigel invasion ( d ) assays indicating the anchorage-independent growth and invasion of MKN-45 and AGS cells stably transfected with mock, circ-hnRNPU , sh-Scb, or sh- circ-hnRNPU #1, and those co-transfected with NONO , c-Myc , sh-NONO #1, or sh-c-Myc #1. Student’s t test compared the difference in c and d . * P < 0.05. Data are shown as mean ± s.e.m. (error bars) or representative of three independent experiments in a - d

Article Snippet: The GST- or Flag-tagged NONO protein and biotin-labeled circular probe of circ-hnRNPU were pulled down by using Flag or GST beads (Sigma).

Techniques: Activity Assay, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, shRNA

Circ-hnRNPU inhibits gastric cancer progression by repressing NONO activity in vivo. a and b , Representative imagines ( a ), in vivo growth curve ( b , left panel), and weight at the end points ( b , right panel) of xenograft tumors formed by subcutaneous injection of AGS cells stably transfected with empty vector (mock) or NONO , and those co-transfected with circ-hnRNPU into the dorsal flanks of nude mice ( n = 5 for each group). c , Representative images (upper panel) and quantification (lower panel) of immunohistochemical staining showing the expression of Ki-67 and CD31 (arrowheads) within xenograft tumors formed by hypodermic injection of AGS cells stably transfected with mock or NONO , and those co-transfected with circ-hnRNPU ( n = 5 for each group). Scale bars: 50 μm. d and e , Western blot (d) and real-time qRT-PCR (e, normalized to β-actin ) assays indicating the levels of NONO , GALNT2 , GALNT6 , MGAT1 , and hnRNPU in xenograft tumors formed by hypodermic injection of AGS cells stably transfected with mock or NONO , and those co-transfected with circ-hnRNPU ( n = 5 for each group). f , Representative images (upper panel), H&E staining (arrowheads), and quantification of lung metastatic colonization (middle panels), as well as Kaplan–Meier curves (lower panel) of nude mice treated with tail vein injection of AGS cells stably transfected with mock or NONO , and those co-transfected with circ-hnRNPU ( n = 4 for each group). Scale bar: 100 μm. Analysis of variance or Student’s t test compared the difference in b , c , e , and f . Log-rank test for survival comparison in f . * P < 0.05, ** P < 0.01. Data are shown as mean ± s.e.m. (error bars) in b , c , e , and f

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circ-hnRNPU inhibits NONO-mediated c-Myc transactivation and mRNA stabilization essential for glycosylation and cancer progression

doi: 10.1186/s13046-023-02898-5

Figure Lengend Snippet: Circ-hnRNPU inhibits gastric cancer progression by repressing NONO activity in vivo. a and b , Representative imagines ( a ), in vivo growth curve ( b , left panel), and weight at the end points ( b , right panel) of xenograft tumors formed by subcutaneous injection of AGS cells stably transfected with empty vector (mock) or NONO , and those co-transfected with circ-hnRNPU into the dorsal flanks of nude mice ( n = 5 for each group). c , Representative images (upper panel) and quantification (lower panel) of immunohistochemical staining showing the expression of Ki-67 and CD31 (arrowheads) within xenograft tumors formed by hypodermic injection of AGS cells stably transfected with mock or NONO , and those co-transfected with circ-hnRNPU ( n = 5 for each group). Scale bars: 50 μm. d and e , Western blot (d) and real-time qRT-PCR (e, normalized to β-actin ) assays indicating the levels of NONO , GALNT2 , GALNT6 , MGAT1 , and hnRNPU in xenograft tumors formed by hypodermic injection of AGS cells stably transfected with mock or NONO , and those co-transfected with circ-hnRNPU ( n = 5 for each group). f , Representative images (upper panel), H&E staining (arrowheads), and quantification of lung metastatic colonization (middle panels), as well as Kaplan–Meier curves (lower panel) of nude mice treated with tail vein injection of AGS cells stably transfected with mock or NONO , and those co-transfected with circ-hnRNPU ( n = 4 for each group). Scale bar: 100 μm. Analysis of variance or Student’s t test compared the difference in b , c , e , and f . Log-rank test for survival comparison in f . * P < 0.05, ** P < 0.01. Data are shown as mean ± s.e.m. (error bars) in b , c , e , and f

Article Snippet: The GST- or Flag-tagged NONO protein and biotin-labeled circular probe of circ-hnRNPU were pulled down by using Flag or GST beads (Sigma).

Techniques: Activity Assay, In Vivo, Injection, Stable Transfection, Transfection, Plasmid Preparation, Immunohistochemistry, Staining, Expressing, Western Blot, Quantitative RT-PCR, Comparison

Therapeutic effects of lentivirus-mediated circ-hnRNPU over-expression in vivo. a , Representative images (left panels), in vivo growth curve (upper middle panel), weight at the end points (upper right panel), and immunohistochemical staining of Ki-67 and CD31 (lower right panels, arrowheads) of xenograft tumors formed by subcutaneous injection of MKN-45 cells into dorsal flanks of nude mice ( n = 5 for each group) that received intravenous administration of lentiviral empty vector (LV-mock) or circ-hnRNPU (LV-circ-hnRNPU) as indicated. Scale bar: 100 μm. b , Representative images (left panel), quantification of lung metastatic colonization (middle panel), and Kaplan–Meier curves (right panel) of nude mice treated with tail vein injection of MKN-45 cells and LV-mock or circ-hnRNPU (LV-circ-hnRNPU) as indicated. Scale bar: 100 μm. c , Real-time qRT-PCR (normalized to β-actin ) assay indicating the levels of NONO , c-Myc , and target genes in normal gastric mucosa ( n = 40) and gastric cancer tissues ( n = 81). d , Kaplan–Meier curves showing the overall survival of gastric cancer cases derived from KM Plotter database ( http://kmplot.com/analysis ) with low or high expression of NONO (cutoff value = 10.31), c-Myc (cutoff value = 10.63), GALNT2 (cutoff value = 8.36), GALNT6 (cutoff value = 9.14), MGAT1 (cutoff value = 9.13), or hnRNPU (cutoff value = 5.21). e , The mechanisms underlying tumor suppressive roles of circ-hnRNPU : as a circRNA derived from hnRNPU , circ-hnRNPU directly interacts with NONO to induce its cytoplasmic retention, which restrains nuclear NONO-facilitated c-Myc transactivation or cytoplasmic NONO-enhanced mRNA stability of glycosyltransferases and hnRNPU , resulting in repression of glycosylation and cancer progression. Analysis of variance or Student’s t test compared the difference in a and b . Mann–Whitney U test compared the difference in c . Log-rank test for survival comparison in b and d . * P < 0.05, ** P < 0.01. Data are shown as mean ± s.e.m. (error bars) in a - c

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circ-hnRNPU inhibits NONO-mediated c-Myc transactivation and mRNA stabilization essential for glycosylation and cancer progression

doi: 10.1186/s13046-023-02898-5

Figure Lengend Snippet: Therapeutic effects of lentivirus-mediated circ-hnRNPU over-expression in vivo. a , Representative images (left panels), in vivo growth curve (upper middle panel), weight at the end points (upper right panel), and immunohistochemical staining of Ki-67 and CD31 (lower right panels, arrowheads) of xenograft tumors formed by subcutaneous injection of MKN-45 cells into dorsal flanks of nude mice ( n = 5 for each group) that received intravenous administration of lentiviral empty vector (LV-mock) or circ-hnRNPU (LV-circ-hnRNPU) as indicated. Scale bar: 100 μm. b , Representative images (left panel), quantification of lung metastatic colonization (middle panel), and Kaplan–Meier curves (right panel) of nude mice treated with tail vein injection of MKN-45 cells and LV-mock or circ-hnRNPU (LV-circ-hnRNPU) as indicated. Scale bar: 100 μm. c , Real-time qRT-PCR (normalized to β-actin ) assay indicating the levels of NONO , c-Myc , and target genes in normal gastric mucosa ( n = 40) and gastric cancer tissues ( n = 81). d , Kaplan–Meier curves showing the overall survival of gastric cancer cases derived from KM Plotter database ( http://kmplot.com/analysis ) with low or high expression of NONO (cutoff value = 10.31), c-Myc (cutoff value = 10.63), GALNT2 (cutoff value = 8.36), GALNT6 (cutoff value = 9.14), MGAT1 (cutoff value = 9.13), or hnRNPU (cutoff value = 5.21). e , The mechanisms underlying tumor suppressive roles of circ-hnRNPU : as a circRNA derived from hnRNPU , circ-hnRNPU directly interacts with NONO to induce its cytoplasmic retention, which restrains nuclear NONO-facilitated c-Myc transactivation or cytoplasmic NONO-enhanced mRNA stability of glycosyltransferases and hnRNPU , resulting in repression of glycosylation and cancer progression. Analysis of variance or Student’s t test compared the difference in a and b . Mann–Whitney U test compared the difference in c . Log-rank test for survival comparison in b and d . * P < 0.05, ** P < 0.01. Data are shown as mean ± s.e.m. (error bars) in a - c

Article Snippet: The GST- or Flag-tagged NONO protein and biotin-labeled circular probe of circ-hnRNPU were pulled down by using Flag or GST beads (Sigma).

Techniques: Over Expression, In Vivo, Immunohistochemistry, Staining, Injection, Plasmid Preparation, Quantitative RT-PCR, Derivative Assay, Expressing, MANN-WHITNEY, Comparison

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