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Santa Cruz Biotechnology staining involved antibodies against nono

A Venn diagram overlapping alternatively spliced events (ASEs) genes, differentially expressed genes (DEGs), and <t>NONO</t> ChIP-Seq targets. B , C Co-immunoprecipitation (Co-IP) in HEK293T cells overexpressing NONO-FLAG and HOXA1-HA ( B ; n = 1), and in Day 2 hiPSC-derived differentiated cells with doxycycline-induced HOXA1-HA ( C ; n = 3), using anti-NONO and anti-HA antibodies for immunoprecipitation and detection. D , E Immunofluorescence staining showing <t>nuclear</t> <t>colocalization</t> of overexpressed HOXA1-HA and endogenous NONO in HEK293T cells ( D ) and in Day 2 differentiated cells ( E ). Specificity of the NONO antibody was validated in NONO-KO cells. Scale bar, 15 μm. n = 3. F GST pull-down assay using recombinant GST-NONO (from E. coli ) and HOXA1-HA (from HEK293T lysates). n = 2. G Schematic representation of HOXA1 and NONO protein domains: Poly-His (PH), hexapeptide (HP), homeodomain (HD), RNA recognition motifs (RRM1, RRM2), and Coiled-coil (Coil) domain. H , I Co-IP domain mapping in HEK293T cells. Interaction of NONO with HOXA1-HA truncation mutants, showing diminished binding upon HOXA1-HD deletion (ΔHD) ( H ). Interaction of HOXA1-HA with NONO-FLAG truncation mutants, showing reduced binding upon NONO-RRM2 or Coil domain deletion ( I ). n = 3. J Structural model of the NONO-HOXA1 interface, highlighting key residues (HOXA1: N230, I275, K286; NONO: E278, Q281, R287). K , L Co-IP validation using point mutants in HEK293T cells. Results show disrupted interaction with the HOXA1-K286A (NONO) mutant ( K ) and the NONO-E278A (HOXA1) mutant ( L ) n = 3. M , N Cycloheximide (CHX) chase assay to assess HOXA1-HA stability. Quantification of HOXA1-HA in nuclear lysates of WT and NONO-KO cells ( M ). Quantification of HOXA1-HA in HEK293T cells co-expressing WT or E278A NONO-FLAG ( N ). Protein levels were normalized to LAMIN A/C and expressed relative to the 0 h time point. n = 3. Data are presented as mean values ± SD. n represents independent biological replicates. Source data are provided as a Source Data file.

Staining Involved Antibodies Against Nono, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Essential role of NONO-HOXA1-Wnt axis in cardiomyocyte differentiation"

Article Title: Essential role of NONO-HOXA1-Wnt axis in cardiomyocyte differentiation

Journal: Nature Communications

doi: 10.1038/s41467-026-68760-2

A Venn diagram overlapping alternatively spliced events (ASEs) genes, differentially expressed genes (DEGs), and NONO ChIP-Seq targets. B , C Co-immunoprecipitation (Co-IP) in HEK293T cells overexpressing NONO-FLAG and HOXA1-HA ( B ; n = 1), and in Day 2 hiPSC-derived differentiated cells with doxycycline-induced HOXA1-HA ( C ; n = 3), using anti-NONO and anti-HA antibodies for immunoprecipitation and detection. D , E Immunofluorescence staining showing nuclear colocalization of overexpressed HOXA1-HA and endogenous NONO in HEK293T cells ( D ) and in Day 2 differentiated cells ( E ). Specificity of the NONO antibody was validated in NONO-KO cells. Scale bar, 15 μm. n = 3. F GST pull-down assay using recombinant GST-NONO (from E. coli ) and HOXA1-HA (from HEK293T lysates). n = 2. G Schematic representation of HOXA1 and NONO protein domains: Poly-His (PH), hexapeptide (HP), homeodomain (HD), RNA recognition motifs (RRM1, RRM2), and Coiled-coil (Coil) domain. H , I Co-IP domain mapping in HEK293T cells. Interaction of NONO with HOXA1-HA truncation mutants, showing diminished binding upon HOXA1-HD deletion (ΔHD) ( H ). Interaction of HOXA1-HA with NONO-FLAG truncation mutants, showing reduced binding upon NONO-RRM2 or Coil domain deletion ( I ). n = 3. J Structural model of the NONO-HOXA1 interface, highlighting key residues (HOXA1: N230, I275, K286; NONO: E278, Q281, R287). K , L Co-IP validation using point mutants in HEK293T cells. Results show disrupted interaction with the HOXA1-K286A (NONO) mutant ( K ) and the NONO-E278A (HOXA1) mutant ( L ) n = 3. M , N Cycloheximide (CHX) chase assay to assess HOXA1-HA stability. Quantification of HOXA1-HA in nuclear lysates of WT and NONO-KO cells ( M ). Quantification of HOXA1-HA in HEK293T cells co-expressing WT or E278A NONO-FLAG ( N ). Protein levels were normalized to LAMIN A/C and expressed relative to the 0 h time point. n = 3. Data are presented as mean values ± SD. n represents independent biological replicates. Source data are provided as a Source Data file.

Figure Legend Snippet: A Venn diagram overlapping alternatively spliced events (ASEs) genes, differentially expressed genes (DEGs), and NONO ChIP-Seq targets. B , C Co-immunoprecipitation (Co-IP) in HEK293T cells overexpressing NONO-FLAG and HOXA1-HA ( B ; n = 1), and in Day 2 hiPSC-derived differentiated cells with doxycycline-induced HOXA1-HA ( C ; n = 3), using anti-NONO and anti-HA antibodies for immunoprecipitation and detection. D , E Immunofluorescence staining showing nuclear colocalization of overexpressed HOXA1-HA and endogenous NONO in HEK293T cells ( D ) and in Day 2 differentiated cells ( E ). Specificity of the NONO antibody was validated in NONO-KO cells. Scale bar, 15 μm. n = 3. F GST pull-down assay using recombinant GST-NONO (from E. coli ) and HOXA1-HA (from HEK293T lysates). n = 2. G Schematic representation of HOXA1 and NONO protein domains: Poly-His (PH), hexapeptide (HP), homeodomain (HD), RNA recognition motifs (RRM1, RRM2), and Coiled-coil (Coil) domain. H , I Co-IP domain mapping in HEK293T cells. Interaction of NONO with HOXA1-HA truncation mutants, showing diminished binding upon HOXA1-HD deletion (ΔHD) ( H ). Interaction of HOXA1-HA with NONO-FLAG truncation mutants, showing reduced binding upon NONO-RRM2 or Coil domain deletion ( I ). n = 3. J Structural model of the NONO-HOXA1 interface, highlighting key residues (HOXA1: N230, I275, K286; NONO: E278, Q281, R287). K , L Co-IP validation using point mutants in HEK293T cells. Results show disrupted interaction with the HOXA1-K286A (NONO) mutant ( K ) and the NONO-E278A (HOXA1) mutant ( L ) n = 3. M , N Cycloheximide (CHX) chase assay to assess HOXA1-HA stability. Quantification of HOXA1-HA in nuclear lysates of WT and NONO-KO cells ( M ). Quantification of HOXA1-HA in HEK293T cells co-expressing WT or E278A NONO-FLAG ( N ). Protein levels were normalized to LAMIN A/C and expressed relative to the 0 h time point. n = 3. Data are presented as mean values ± SD. n represents independent biological replicates. Source data are provided as a Source Data file.

Techniques Used: ChIP-sequencing, Immunoprecipitation, Co-Immunoprecipitation Assay, Derivative Assay, Immunofluorescence, Staining, Pull Down Assay, Recombinant, Binding Assay, Biomarker Discovery, Mutagenesis, Expressing

A Genome browser visualization of ChIP-seq tracks at key cardiac gene loci. B GO biological process enrichment analysis of selected terms for genes bound by NONO in Day 2 differentiated Cells. C Venn diagram of NONO-enriched and HOXA1-HA enriched overlap peaks in Day 2 differentiated cells. D GO biological process enrichment analysis of selected terms for genes of NONO and HOXA1-HA co-binding. E Heatmaps of HOXA1 ChIP-seq signals in WT and NONO-KO cells. HOXA1 binding is significantly reduced in the absence of NONO, with shared and lost peaks shown. F Representative ChIP-seq tracks of HOXA1 binding at precardiac mesoderm loci ( MESP1 and PDGFRA ) in Day 2 WT and NONO-KO cells. G ChIP-qPCR analysis showing reduced HOXA1 binding at target loci in Day 2 differentiated NONO-KO cells compared to WT. n = 3. H GO enrichment analysis of genes with lost HOXA1 binding in NONO-KO cells and differential expression (DEGs) in HOXA1-KO cells. I qRT-PCR analysis of HOXA1-regulated genes in Day 2 WT, NONO-KO, and HOXA1-KO cells. n = 3. J Co-immunoprecipitation (Co-IP) assessment of NONO homodimerization in Day 2 cells expressing Dox-induced NONO-FLAG and NONO-HA. n = 3. K , L Co-IP analysis of HOXA1 homodimerization in Day 2 WT and NONO-KO cells co-expressing HOXA1-HA and HOXA1-FLAG, using anti-HA ( K ) and anti-FLAG ( L ) antibodies for immunoprecipitation, followed by immunoblotting with anti-FLAG and anti-HA antibodies. Note the impaired HOXA1 self-association in NONO-KO cells. n = 3. M , N Flow cytometry analysis ( M ) and quantification ( N ) of cTNT-positive cardiomyocytes in NONO-KO and NONO/HOXA1 Double-KO (DKO) at Day 15. n = 3. O , P Immunostaining ( O ) and quantification ( P ) of sarcomere organization (α-ACTININ, green; cTNT, red) in Day 15 NONO-KO and DKO cardiomyocytes. Scale bar, 15 μm. ( n = 4; NONO-KO, 27 cells; DKO, 30 cells). Data are presented as mean values ± SD. P values were calculated using a two-tailed unpaired Student’s t test, except for ( P ) (Fisher’s exact test). P < 0.05 was considered significant. n represents independent biological replicates. Source data are provided as a Source Data file.

Figure Legend Snippet: A Genome browser visualization of ChIP-seq tracks at key cardiac gene loci. B GO biological process enrichment analysis of selected terms for genes bound by NONO in Day 2 differentiated Cells. C Venn diagram of NONO-enriched and HOXA1-HA enriched overlap peaks in Day 2 differentiated cells. D GO biological process enrichment analysis of selected terms for genes of NONO and HOXA1-HA co-binding. E Heatmaps of HOXA1 ChIP-seq signals in WT and NONO-KO cells. HOXA1 binding is significantly reduced in the absence of NONO, with shared and lost peaks shown. F Representative ChIP-seq tracks of HOXA1 binding at precardiac mesoderm loci ( MESP1 and PDGFRA ) in Day 2 WT and NONO-KO cells. G ChIP-qPCR analysis showing reduced HOXA1 binding at target loci in Day 2 differentiated NONO-KO cells compared to WT. n = 3. H GO enrichment analysis of genes with lost HOXA1 binding in NONO-KO cells and differential expression (DEGs) in HOXA1-KO cells. I qRT-PCR analysis of HOXA1-regulated genes in Day 2 WT, NONO-KO, and HOXA1-KO cells. n = 3. J Co-immunoprecipitation (Co-IP) assessment of NONO homodimerization in Day 2 cells expressing Dox-induced NONO-FLAG and NONO-HA. n = 3. K , L Co-IP analysis of HOXA1 homodimerization in Day 2 WT and NONO-KO cells co-expressing HOXA1-HA and HOXA1-FLAG, using anti-HA ( K ) and anti-FLAG ( L ) antibodies for immunoprecipitation, followed by immunoblotting with anti-FLAG and anti-HA antibodies. Note the impaired HOXA1 self-association in NONO-KO cells. n = 3. M , N Flow cytometry analysis ( M ) and quantification ( N ) of cTNT-positive cardiomyocytes in NONO-KO and NONO/HOXA1 Double-KO (DKO) at Day 15. n = 3. O , P Immunostaining ( O ) and quantification ( P ) of sarcomere organization (α-ACTININ, green; cTNT, red) in Day 15 NONO-KO and DKO cardiomyocytes. Scale bar, 15 μm. ( n = 4; NONO-KO, 27 cells; DKO, 30 cells). Data are presented as mean values ± SD. P values were calculated using a two-tailed unpaired Student’s t test, except for ( P ) (Fisher’s exact test). P < 0.05 was considered significant. n represents independent biological replicates. Source data are provided as a Source Data file.

Techniques Used: ChIP-sequencing, Binding Assay, ChIP-qPCR, Quantitative Proteomics, Quantitative RT-PCR, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Western Blot, Flow Cytometry, Immunostaining, Two Tailed Test

A Heatmap showing log2-fold changes of differentially expressed genes (DEGs) among NONO-dependent and HOXA1-regulated WNT pathway genes in NONO-KO (left) and HOXA1-KO (right) cells. B Representative ChIP-seq tracks of WNT pathway genes showing HOXA1 binding in Day 2 WT cells, with reduced binding in NONO-KO cells. C , D Western blot analysis of cytoplasmic and nuclear β-CATENIN levels in Day 2 differentiated cells. WT vs. HOXA1-KO ( C ). WT, NONO-KO, and NONO-RE ( D ). n = 3. E , F Cycloheximide (CHX) chase assays and quantification of nuclear β-catenin stability in NONO-KO ( E ) and HOXA1-KO ( F ) cells. Protein levels were normalized to LAMIN A/C and expressed relative to the 0 h time point. n = 3. G – J TOP/FOP reporter assays in HEK293T cells treated with or without CHIR99021 (CHIR). Dose-dependent knockdown of HOXA1 ( G ) (siHOXA1; n = 5). Dose-dependent overexpression of HOXA1 ( H ) ( n = 5). Dose-dependent knockdown of NONO ( I ) (siNONO; n = 6). Dose-dependent overexpression of NONO ( J ) ( n = 5). K TOP/FOP reporter assay in cells overexpressing full-length HOXA1 or homeodomain-deleted mutant (HOXA1 ΔHD), treated with or without CHIR. n = 4. L TOP/FOP reporter assay in cells expressing WT or K286A mutants (MU) HOXA1, followed by NONO overexpression. Cells were treated with or without CHIR. n = 5. M , N Flow cytometry analysis ( M ) and quantification ( N ) of cTNT + cardiomyocytes in Day 15 NONO-KO cultures treated with CHIR (8–14 μM) during Days 0–2. n = 3. O qRT-PCR analysis of TNNT2 , MYH6 , and MYH7 expression in Day 15 NONO-KO cardiomyocytes treated with CHIR (8–14 μM) during Days 0–2. n = 3. P Schematic model of the NONO-HOXA1-Wnt signaling axis in cardiomyocyte differentiation. Data are presented as mean values ± SD. P values were calculated using a two-tailed unpaired Student’s t test. P < 0.05 was considered significant. n represents independent biological replicates. Source data are provided as a Source Data file.

Figure Legend Snippet: A Heatmap showing log2-fold changes of differentially expressed genes (DEGs) among NONO-dependent and HOXA1-regulated WNT pathway genes in NONO-KO (left) and HOXA1-KO (right) cells. B Representative ChIP-seq tracks of WNT pathway genes showing HOXA1 binding in Day 2 WT cells, with reduced binding in NONO-KO cells. C , D Western blot analysis of cytoplasmic and nuclear β-CATENIN levels in Day 2 differentiated cells. WT vs. HOXA1-KO ( C ). WT, NONO-KO, and NONO-RE ( D ). n = 3. E , F Cycloheximide (CHX) chase assays and quantification of nuclear β-catenin stability in NONO-KO ( E ) and HOXA1-KO ( F ) cells. Protein levels were normalized to LAMIN A/C and expressed relative to the 0 h time point. n = 3. G – J TOP/FOP reporter assays in HEK293T cells treated with or without CHIR99021 (CHIR). Dose-dependent knockdown of HOXA1 ( G ) (siHOXA1; n = 5). Dose-dependent overexpression of HOXA1 ( H ) ( n = 5). Dose-dependent knockdown of NONO ( I ) (siNONO; n = 6). Dose-dependent overexpression of NONO ( J ) ( n = 5). K TOP/FOP reporter assay in cells overexpressing full-length HOXA1 or homeodomain-deleted mutant (HOXA1 ΔHD), treated with or without CHIR. n = 4. L TOP/FOP reporter assay in cells expressing WT or K286A mutants (MU) HOXA1, followed by NONO overexpression. Cells were treated with or without CHIR. n = 5. M , N Flow cytometry analysis ( M ) and quantification ( N ) of cTNT + cardiomyocytes in Day 15 NONO-KO cultures treated with CHIR (8–14 μM) during Days 0–2. n = 3. O qRT-PCR analysis of TNNT2 , MYH6 , and MYH7 expression in Day 15 NONO-KO cardiomyocytes treated with CHIR (8–14 μM) during Days 0–2. n = 3. P Schematic model of the NONO-HOXA1-Wnt signaling axis in cardiomyocyte differentiation. Data are presented as mean values ± SD. P values were calculated using a two-tailed unpaired Student’s t test. P < 0.05 was considered significant. n represents independent biological replicates. Source data are provided as a Source Data file.

Techniques Used: ChIP-sequencing, Binding Assay, Western Blot, Knockdown, Over Expression, Reporter Assay, Mutagenesis, Expressing, Flow Cytometry, Quantitative RT-PCR, Two Tailed Test

Image Search Results

Journal: Nature Communications

Article Title: Essential role of NONO-HOXA1-Wnt axis in cardiomyocyte differentiation

doi: 10.1038/s41467-026-68760-2

Figure Lengend Snippet: A Venn diagram overlapping alternatively spliced events (ASEs) genes, differentially expressed genes (DEGs), and NONO ChIP-Seq targets. B , C Co-immunoprecipitation (Co-IP) in HEK293T cells overexpressing NONO-FLAG and HOXA1-HA ( B ; n = 1), and in Day 2 hiPSC-derived differentiated cells with doxycycline-induced HOXA1-HA ( C ; n = 3), using anti-NONO and anti-HA antibodies for immunoprecipitation and detection. D , E Immunofluorescence staining showing nuclear colocalization of overexpressed HOXA1-HA and endogenous NONO in HEK293T cells ( D ) and in Day 2 differentiated cells ( E ). Specificity of the NONO antibody was validated in NONO-KO cells. Scale bar, 15 μm. n = 3. F GST pull-down assay using recombinant GST-NONO (from E. coli ) and HOXA1-HA (from HEK293T lysates). n = 2. G Schematic representation of HOXA1 and NONO protein domains: Poly-His (PH), hexapeptide (HP), homeodomain (HD), RNA recognition motifs (RRM1, RRM2), and Coiled-coil (Coil) domain. H , I Co-IP domain mapping in HEK293T cells. Interaction of NONO with HOXA1-HA truncation mutants, showing diminished binding upon HOXA1-HD deletion (ΔHD) ( H ). Interaction of HOXA1-HA with NONO-FLAG truncation mutants, showing reduced binding upon NONO-RRM2 or Coil domain deletion ( I ). n = 3. J Structural model of the NONO-HOXA1 interface, highlighting key residues (HOXA1: N230, I275, K286; NONO: E278, Q281, R287). K , L Co-IP validation using point mutants in HEK293T cells. Results show disrupted interaction with the HOXA1-K286A (NONO) mutant ( K ) and the NONO-E278A (HOXA1) mutant ( L ) n = 3. M , N Cycloheximide (CHX) chase assay to assess HOXA1-HA stability. Quantification of HOXA1-HA in nuclear lysates of WT and NONO-KO cells ( M ). Quantification of HOXA1-HA in HEK293T cells co-expressing WT or E278A NONO-FLAG ( N ). Protein levels were normalized to LAMIN A/C and expressed relative to the 0 h time point. n = 3. Data are presented as mean values ± SD. n represents independent biological replicates. Source data are provided as a Source Data file.

Article Snippet: For colocalization studies of NONO and HOXA1, staining involved antibodies against NONO (Santa Cruz, sc-376865) at a 1:200 dilution and HA tag (Abcam, ab9110) at a 1:500 dilution.

Techniques: ChIP-sequencing, Immunoprecipitation, Co-Immunoprecipitation Assay, Derivative Assay, Immunofluorescence, Staining, Pull Down Assay, Recombinant, Binding Assay, Biomarker Discovery, Mutagenesis, Expressing

Journal: Nature Communications

Article Title: Essential role of NONO-HOXA1-Wnt axis in cardiomyocyte differentiation

doi: 10.1038/s41467-026-68760-2

Figure Lengend Snippet: A Genome browser visualization of ChIP-seq tracks at key cardiac gene loci. B GO biological process enrichment analysis of selected terms for genes bound by NONO in Day 2 differentiated Cells. C Venn diagram of NONO-enriched and HOXA1-HA enriched overlap peaks in Day 2 differentiated cells. D GO biological process enrichment analysis of selected terms for genes of NONO and HOXA1-HA co-binding. E Heatmaps of HOXA1 ChIP-seq signals in WT and NONO-KO cells. HOXA1 binding is significantly reduced in the absence of NONO, with shared and lost peaks shown. F Representative ChIP-seq tracks of HOXA1 binding at precardiac mesoderm loci ( MESP1 and PDGFRA ) in Day 2 WT and NONO-KO cells. G ChIP-qPCR analysis showing reduced HOXA1 binding at target loci in Day 2 differentiated NONO-KO cells compared to WT. n = 3. H GO enrichment analysis of genes with lost HOXA1 binding in NONO-KO cells and differential expression (DEGs) in HOXA1-KO cells. I qRT-PCR analysis of HOXA1-regulated genes in Day 2 WT, NONO-KO, and HOXA1-KO cells. n = 3. J Co-immunoprecipitation (Co-IP) assessment of NONO homodimerization in Day 2 cells expressing Dox-induced NONO-FLAG and NONO-HA. n = 3. K , L Co-IP analysis of HOXA1 homodimerization in Day 2 WT and NONO-KO cells co-expressing HOXA1-HA and HOXA1-FLAG, using anti-HA ( K ) and anti-FLAG ( L ) antibodies for immunoprecipitation, followed by immunoblotting with anti-FLAG and anti-HA antibodies. Note the impaired HOXA1 self-association in NONO-KO cells. n = 3. M , N Flow cytometry analysis ( M ) and quantification ( N ) of cTNT-positive cardiomyocytes in NONO-KO and NONO/HOXA1 Double-KO (DKO) at Day 15. n = 3. O , P Immunostaining ( O ) and quantification ( P ) of sarcomere organization (α-ACTININ, green; cTNT, red) in Day 15 NONO-KO and DKO cardiomyocytes. Scale bar, 15 μm. ( n = 4; NONO-KO, 27 cells; DKO, 30 cells). Data are presented as mean values ± SD. P values were calculated using a two-tailed unpaired Student’s t test, except for ( P ) (Fisher’s exact test). P < 0.05 was considered significant. n represents independent biological replicates. Source data are provided as a Source Data file.

Techniques: ChIP-sequencing, Binding Assay, ChIP-qPCR, Quantitative Proteomics, Quantitative RT-PCR, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Western Blot, Flow Cytometry, Immunostaining, Two Tailed Test

Journal: Nature Communications

Article Title: Essential role of NONO-HOXA1-Wnt axis in cardiomyocyte differentiation

doi: 10.1038/s41467-026-68760-2

Figure Lengend Snippet: A Heatmap showing log2-fold changes of differentially expressed genes (DEGs) among NONO-dependent and HOXA1-regulated WNT pathway genes in NONO-KO (left) and HOXA1-KO (right) cells. B Representative ChIP-seq tracks of WNT pathway genes showing HOXA1 binding in Day 2 WT cells, with reduced binding in NONO-KO cells. C , D Western blot analysis of cytoplasmic and nuclear β-CATENIN levels in Day 2 differentiated cells. WT vs. HOXA1-KO ( C ). WT, NONO-KO, and NONO-RE ( D ). n = 3. E , F Cycloheximide (CHX) chase assays and quantification of nuclear β-catenin stability in NONO-KO ( E ) and HOXA1-KO ( F ) cells. Protein levels were normalized to LAMIN A/C and expressed relative to the 0 h time point. n = 3. G – J TOP/FOP reporter assays in HEK293T cells treated with or without CHIR99021 (CHIR). Dose-dependent knockdown of HOXA1 ( G ) (siHOXA1; n = 5). Dose-dependent overexpression of HOXA1 ( H ) ( n = 5). Dose-dependent knockdown of NONO ( I ) (siNONO; n = 6). Dose-dependent overexpression of NONO ( J ) ( n = 5). K TOP/FOP reporter assay in cells overexpressing full-length HOXA1 or homeodomain-deleted mutant (HOXA1 ΔHD), treated with or without CHIR. n = 4. L TOP/FOP reporter assay in cells expressing WT or K286A mutants (MU) HOXA1, followed by NONO overexpression. Cells were treated with or without CHIR. n = 5. M , N Flow cytometry analysis ( M ) and quantification ( N ) of cTNT + cardiomyocytes in Day 15 NONO-KO cultures treated with CHIR (8–14 μM) during Days 0–2. n = 3. O qRT-PCR analysis of TNNT2 , MYH6 , and MYH7 expression in Day 15 NONO-KO cardiomyocytes treated with CHIR (8–14 μM) during Days 0–2. n = 3. P Schematic model of the NONO-HOXA1-Wnt signaling axis in cardiomyocyte differentiation. Data are presented as mean values ± SD. P values were calculated using a two-tailed unpaired Student’s t test. P < 0.05 was considered significant. n represents independent biological replicates. Source data are provided as a Source Data file.

Techniques: ChIP-sequencing, Binding Assay, Western Blot, Knockdown, Over Expression, Reporter Assay, Mutagenesis, Expressing, Flow Cytometry, Quantitative RT-PCR, Two Tailed Test

DNA damage enhances the association between NONO and OGT. A Left panel: IR induces O-GlcNAcylation of NONO. HEK293T cells were exposed to 0, 2, 5 or 10 Gy of IR. Following treatment, cells were lysed using NETN300 buffer, and immunoprecipitation (IP) and Western blot were performed with the indicated antibodies. Right panel: Quantification of NONO O-GlcNAcylation levels, normalized to NONO levels and presented as fold change relative to control samples ( n = 3 per condition). B , C Investigation of the exogenous interaction between OGT and NONO via Co-IP. HEK293T cells were transfected with SFB-NONO and Myc-OGT plasmids. IP followed by Western blot was conducted using the indicated antibodies to assess the interaction between OGT and NONO. D Left panel: DNA damage enhances the endogenous interaction between NONO and OGT. HEK293T cells were exposed to 10 Gy of IR and lysed with NETN300 buffer. Cell extracts were subjected to IP and Western blot analysis using the indicated antibodies. Right panel: Quantification of OGT level, normalized to NONO levels and presented as fold change relative to control samples ( n = 3 per condition)

Journal: Genome Biology

Article Title: O-GlcNAcylation of NONO mediates alternative splicing of SETMAR and facilitates NHEJ repair

doi: 10.1186/s13059-026-03930-5

Figure Lengend Snippet: DNA damage enhances the association between NONO and OGT. A Left panel: IR induces O-GlcNAcylation of NONO. HEK293T cells were exposed to 0, 2, 5 or 10 Gy of IR. Following treatment, cells were lysed using NETN300 buffer, and immunoprecipitation (IP) and Western blot were performed with the indicated antibodies. Right panel: Quantification of NONO O-GlcNAcylation levels, normalized to NONO levels and presented as fold change relative to control samples ( n = 3 per condition). B , C Investigation of the exogenous interaction between OGT and NONO via Co-IP. HEK293T cells were transfected with SFB-NONO and Myc-OGT plasmids. IP followed by Western blot was conducted using the indicated antibodies to assess the interaction between OGT and NONO. D Left panel: DNA damage enhances the endogenous interaction between NONO and OGT. HEK293T cells were exposed to 10 Gy of IR and lysed with NETN300 buffer. Cell extracts were subjected to IP and Western blot analysis using the indicated antibodies. Right panel: Quantification of OGT level, normalized to NONO levels and presented as fold change relative to control samples ( n = 3 per condition)

Article Snippet: Antibodies used in this study include the following: Anti-DDDDK-tag mAb (MBL, M185-3L), Anti-Myc-tag mAb (MBL, M047-3), Anti-GAPDH (Proteintech, 60,004–1-Ig), OGT polyclonal antibody (Proteintech, 11,576–2-AP), NONO polyclonal antibody (Proteintech, 11,058–1-AP), RL2 (Santa cruz biotechnology, SC-59624, which is an antibody against O-GlcNAc), Histone-H3 polyclonal antibody (Proteintech, 17,168–1-AP), anti-HA (Sigma, 66,006–2-Ig), Anti-PAR monoclonal antibody (R&D, 4335-MC-100-AC), Ku80 polyclonal antibody (Proteintech, 16,389–1-AP), SFPQ polyclonal antibody (Proteintech, 15,585–1-AP), anti-Histone H3(di-methyl K36) polyclonal antibody (abcam, AB9049), Ku70 polyclonal antibody (Proteintech, 10,723–1-AP), anti-SETMAR polyclonal antibody (abcam, ab129455), anti-RAD51 monoclonal antibody(abcam, ab133534).

Techniques: Immunoprecipitation, Western Blot, Control, Co-Immunoprecipitation Assay, Transfection

$O-GlcNAcylation enhances the stability of NONO protein. A Left panel: O-GlcNAcylation promotes NONO recruitment to DNA damage sites. U2OS cells expressing GFP-NONO were subjected to laser microirradiation, with or without OSMI-1 treatment. Scale bar: 5 μm. Right panel: Quantification of GFP-NONO intensity at laser-induced damage sites. The intensity of GFP-NONO at laser stripes was quantified at indicated time points using Image J, and peak fluorescence density in the micro-irradiated areas was plotted against time. B Left panel: Inhibition of O-GlcNAcylation reduces NONO binding to chromatin. HEK293T cells were transfected with SFB-NONO and treated with either DMSO or 20 μM OSMI-1 for 48 h. Whole cell extract (WCE), non-chromatin, and chromatin fractions of cells were harvested and analyzed by immunoblotting with the indicated antibodies. Right panel: Quantification of Flag (NONO) level, normalized to histone H3 levels in chromatin and presented as fold change relative to control samples ( n = 3 per condition). C Left panel: Time-dependent degradation of NONO upon OGT inhibition. HEK293T cells were treated with 20 μM OSMI-1 or mock treatment for up to 72 h. NONO protein levels were assessed using Western blot, with GAPDH serving as the loading control. Right panel: Quantification of NONO level, normalized to GAPDH levels and presented as fold change relative to control samples ( n = 3 per condition). D Left panel: Inhibition of O-GlcNAcylation accelerates NONO degradation. HEK293T cells were treated with either DMSO or 20 μM OSMI-1 for 48 h, followed by 50 μM CHX treatment for up to 8 h. NONO protein levels were monitored using Western blot analysis, with GAPDH as the loading control. Right panel: Quantification of NONO level, normalized to GAPDH and presented as fold change relative to control samples ( n = 3 per condition)$

Journal: Genome Biology

Article Title: O-GlcNAcylation of NONO mediates alternative splicing of SETMAR and facilitates NHEJ repair

doi: 10.1186/s13059-026-03930-5

Figure Lengend Snippet: O-GlcNAcylation enhances the stability of NONO protein. A Left panel: O-GlcNAcylation promotes NONO recruitment to DNA damage sites. U2OS cells expressing GFP-NONO were subjected to laser microirradiation, with or without OSMI-1 treatment. Scale bar: 5 μm. Right panel: Quantification of GFP-NONO intensity at laser-induced damage sites. The intensity of GFP-NONO at laser stripes was quantified at indicated time points using Image J, and peak fluorescence density in the micro-irradiated areas was plotted against time. B Left panel: Inhibition of O-GlcNAcylation reduces NONO binding to chromatin. HEK293T cells were transfected with SFB-NONO and treated with either DMSO or 20 μM OSMI-1 for 48 h. Whole cell extract (WCE), non-chromatin, and chromatin fractions of cells were harvested and analyzed by immunoblotting with the indicated antibodies. Right panel: Quantification of Flag (NONO) level, normalized to histone H3 levels in chromatin and presented as fold change relative to control samples ( n = 3 per condition). C Left panel: Time-dependent degradation of NONO upon OGT inhibition. HEK293T cells were treated with 20 μM OSMI-1 or mock treatment for up to 72 h. NONO protein levels were assessed using Western blot, with GAPDH serving as the loading control. Right panel: Quantification of NONO level, normalized to GAPDH levels and presented as fold change relative to control samples ( n = 3 per condition). D Left panel: Inhibition of O-GlcNAcylation accelerates NONO degradation. HEK293T cells were treated with either DMSO or 20 μM OSMI-1 for 48 h, followed by 50 μM CHX treatment for up to 8 h. NONO protein levels were monitored using Western blot analysis, with GAPDH as the loading control. Right panel: Quantification of NONO level, normalized to GAPDH and presented as fold change relative to control samples ( n = 3 per condition)

Techniques: Expressing, Fluorescence, Irradiation, Inhibition, Binding Assay, Transfection, Western Blot, Control

$O-GlcNAcylation at S147 is required for NONO-mediated DNA damage repair. A Assessment of NONO O-GlcNAcylation. HEK293T cells were transfected with empty vector (EV), SFB-NONO-WT, S147A, T440A or 2 A plasmids, then treated by 10 Gy of IR. Cell lysates were denatured, immunoprecipitated with streptavidin beads, and analyzed via immunoblotting with O-GlcNAc and Flag antibodies. B Conservation of the S147 locus across different species. The upper panel depicts wild-type NONO, including RNA recognition motifs (RRM1/2), the NOPS domain, coiled-coil domain (CC), and C-terminal intrinsically disordered domains. The lower panel shows a BLAST alignment of the S147 locus across species. C Left panel: O-GlcNAcylation of Ser147 facilitates NONO recruitment to DNA damage sites. U2OS cells transfected with GFP-NONO-WT or S147A were subjected to laser microirradiation. Scale bar: 5 μm. Right panel: Quantification of GFP-NONO intensity at laser strips using Image J, and peak fluorescence density in the micro-irradiated areas was plotted against time. D Left panel: S147A exhibited impaired chromatin-binding without MG132 treatment. HEK293T cells transfected with SFB-NONO-WT or S147A were subjected to fraction extraction and immunoblotting. Right panel: Quantification of Flag (NONO) levels ( n = 3 per condition). E Left panel: O-GlcNAcylation of S147 is essential for NONO-mediated DNA damage repair. Stable HEK293T cells with NONO knockdown and reconstituted WT or S147A were exposed to 10 Gy of IR and underwent neutral comet assays (* P < 0.05). Right panel: Quantitative analysis of comet assay results from three independent experiments (50 cells per time point). Data are presented as mean ± SEM. (F) O-GlcNAcylation of Ser147 promotes NONO-mediated NHEJ repair. NONO knockdown HEK293T cells reconstituted WT or S147A were transfected with an NHEJ reporter pre-digested by Hind III and cultured for 48 h. FACS analysis quantified GFP-positive cells. Data from three independent experiments are presented as mean ± SD. ** P < 0.01, *** P < 0.001. Stable HEK293T cells with Ku80 knockdown were used as a positive control$

Journal: Genome Biology

Article Title: O-GlcNAcylation of NONO mediates alternative splicing of SETMAR and facilitates NHEJ repair

doi: 10.1186/s13059-026-03930-5

Figure Lengend Snippet: O-GlcNAcylation at S147 is required for NONO-mediated DNA damage repair. A Assessment of NONO O-GlcNAcylation. HEK293T cells were transfected with empty vector (EV), SFB-NONO-WT, S147A, T440A or 2 A plasmids, then treated by 10 Gy of IR. Cell lysates were denatured, immunoprecipitated with streptavidin beads, and analyzed via immunoblotting with O-GlcNAc and Flag antibodies. B Conservation of the S147 locus across different species. The upper panel depicts wild-type NONO, including RNA recognition motifs (RRM1/2), the NOPS domain, coiled-coil domain (CC), and C-terminal intrinsically disordered domains. The lower panel shows a BLAST alignment of the S147 locus across species. C Left panel: O-GlcNAcylation of Ser147 facilitates NONO recruitment to DNA damage sites. U2OS cells transfected with GFP-NONO-WT or S147A were subjected to laser microirradiation. Scale bar: 5 μm. Right panel: Quantification of GFP-NONO intensity at laser strips using Image J, and peak fluorescence density in the micro-irradiated areas was plotted against time. D Left panel: S147A exhibited impaired chromatin-binding without MG132 treatment. HEK293T cells transfected with SFB-NONO-WT or S147A were subjected to fraction extraction and immunoblotting. Right panel: Quantification of Flag (NONO) levels ( n = 3 per condition). E Left panel: O-GlcNAcylation of S147 is essential for NONO-mediated DNA damage repair. Stable HEK293T cells with NONO knockdown and reconstituted WT or S147A were exposed to 10 Gy of IR and underwent neutral comet assays (* P < 0.05). Right panel: Quantitative analysis of comet assay results from three independent experiments (50 cells per time point). Data are presented as mean ± SEM. (F) O-GlcNAcylation of Ser147 promotes NONO-mediated NHEJ repair. NONO knockdown HEK293T cells reconstituted WT or S147A were transfected with an NHEJ reporter pre-digested by Hind III and cultured for 48 h. FACS analysis quantified GFP-positive cells. Data from three independent experiments are presented as mean ± SD. ** P < 0.01, *** P < 0.001. Stable HEK293T cells with Ku80 knockdown were used as a positive control

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Fluorescence, Irradiation, Binding Assay, Extraction, Knockdown, Single Cell Gel Electrophoresis, Cell Culture, Positive Control

Ser147 O-GlcNAcylation of NONO antagonizes its ubiquitination by decreasing interaction with RNF8. A The proteasome inhibitor MG132 prevents degradation of the NONO S147A mutant. HEK293T cells were transfected with SFB-NONO-WT or the S147A mutant and treated with or without 10 μM MG132. Cell lysates were analyzed by Western blot analysis with the indicated antibodies. B Left panel: O-GlcNAcylation at S147 is critical for NONO stability. HEK293T cells expressing SFB-NONO-WT or S147A were treated with 50 μM CHX for 12 h. Protein levels were monitored by Western blot using anti-FLAG antibodies, with GAPDH serving as the loading control. Right panel: Quantification of Flag (NONO) expression, normalized to GAPDH levels and presented as fold change relative to control samples ( n = 3 per condition). C O-GlcNAcylation of NONO crosstalks with ubiquitination. HEK293T cells were transfected with Myc-RNF8, HA-Ub, and either SFB-NONO WT or the S147A mutant, followed by an in vivo ubiquitination assay. D O-GlcNAcylation of Ser147 impairs the interaction between NONO and RNF8. HEK293T cells were transfected with Myc-RNF8 and either SFB-NONO WT or S147A mutant. The interaction was assessed by IP assay

Journal: Genome Biology

Article Title: O-GlcNAcylation of NONO mediates alternative splicing of SETMAR and facilitates NHEJ repair

doi: 10.1186/s13059-026-03930-5

Figure Lengend Snippet: Ser147 O-GlcNAcylation of NONO antagonizes its ubiquitination by decreasing interaction with RNF8. A The proteasome inhibitor MG132 prevents degradation of the NONO S147A mutant. HEK293T cells were transfected with SFB-NONO-WT or the S147A mutant and treated with or without 10 μM MG132. Cell lysates were analyzed by Western blot analysis with the indicated antibodies. B Left panel: O-GlcNAcylation at S147 is critical for NONO stability. HEK293T cells expressing SFB-NONO-WT or S147A were treated with 50 μM CHX for 12 h. Protein levels were monitored by Western blot using anti-FLAG antibodies, with GAPDH serving as the loading control. Right panel: Quantification of Flag (NONO) expression, normalized to GAPDH levels and presented as fold change relative to control samples ( n = 3 per condition). C O-GlcNAcylation of NONO crosstalks with ubiquitination. HEK293T cells were transfected with Myc-RNF8, HA-Ub, and either SFB-NONO WT or the S147A mutant, followed by an in vivo ubiquitination assay. D O-GlcNAcylation of Ser147 impairs the interaction between NONO and RNF8. HEK293T cells were transfected with Myc-RNF8 and either SFB-NONO WT or S147A mutant. The interaction was assessed by IP assay

Techniques: Ubiquitin Proteomics, Mutagenesis, Transfection, Western Blot, Expressing, Control, In Vivo

O-GlcNAcylation of NONO promotes NHEJ by regulating the alternative splicing of pre-mRNA of SETMAR. A Schematic illustrating how the NONO-SFPQ complex influences NHEJ repair by modulating the alternative splicing of SETMAR precursor mRNA. B O-GlcNAcylation of Ser147 stabilizes the assembly of the NONO-SFPQ complex. HEK293T cells were transfected with either SFB-NONO or the S147A mutant. Cell lysates were immunoprecipitated with Streptavidin beads, followed by Western blotting with anti-SFPQ or anti-Flag antibodies. C Quantification of qPCR analysis of SETMAR pre-mRNA from the RIP assay of HEK293T cells. Cells were transfected with SFB-NONO, S147A mutant, or an empty vector. Cell lysates were subjected to RIP, and the products were amplified by qPCR using the indicated primers pairs. Input was used for normalization, and the empty vector served as a negative control. D Western blot analysis of RNA pull-down eluates using anti-Myc antibodies demonstrates a higher binding specificity of NONO-WT for CAGGCAGG RNA repeats compared to the S147A mutant in HEK293T cells. E Quantification of the SETMAR-L/SETMAR-S ratio in HEK293T cells by qPCR. F Left panel: RNA FISH analysis shows that O-GlcNAcylation of NONO promotes the expression of SETMAR-L. RNA FISH was conducted to visualize SETMAR-L (green) and SETMAR-S (red) in NONO-knockdown HEK293T cells stably reconstituted with either WT NONO or the S147A mutant. DAPI staining of the nuclei is shown in blue. Scale bar, 20 μm. Right panel: Quantification of fluorescence intensity from FISH. Data from three independent experiments are presented as mean ± SD. Scale bar, 20 μm

Journal: Genome Biology

Article Title: O-GlcNAcylation of NONO mediates alternative splicing of SETMAR and facilitates NHEJ repair

doi: 10.1186/s13059-026-03930-5

Figure Lengend Snippet: O-GlcNAcylation of NONO promotes NHEJ by regulating the alternative splicing of pre-mRNA of SETMAR. A Schematic illustrating how the NONO-SFPQ complex influences NHEJ repair by modulating the alternative splicing of SETMAR precursor mRNA. B O-GlcNAcylation of Ser147 stabilizes the assembly of the NONO-SFPQ complex. HEK293T cells were transfected with either SFB-NONO or the S147A mutant. Cell lysates were immunoprecipitated with Streptavidin beads, followed by Western blotting with anti-SFPQ or anti-Flag antibodies. C Quantification of qPCR analysis of SETMAR pre-mRNA from the RIP assay of HEK293T cells. Cells were transfected with SFB-NONO, S147A mutant, or an empty vector. Cell lysates were subjected to RIP, and the products were amplified by qPCR using the indicated primers pairs. Input was used for normalization, and the empty vector served as a negative control. D Western blot analysis of RNA pull-down eluates using anti-Myc antibodies demonstrates a higher binding specificity of NONO-WT for CAGGCAGG RNA repeats compared to the S147A mutant in HEK293T cells. E Quantification of the SETMAR-L/SETMAR-S ratio in HEK293T cells by qPCR. F Left panel: RNA FISH analysis shows that O-GlcNAcylation of NONO promotes the expression of SETMAR-L. RNA FISH was conducted to visualize SETMAR-L (green) and SETMAR-S (red) in NONO-knockdown HEK293T cells stably reconstituted with either WT NONO or the S147A mutant. DAPI staining of the nuclei is shown in blue. Scale bar, 20 μm. Right panel: Quantification of fluorescence intensity from FISH. Data from three independent experiments are presented as mean ± SD. Scale bar, 20 μm

Techniques: Alternative Splicing, Transfection, Mutagenesis, Immunoprecipitation, Western Blot, Plasmid Preparation, Amplification, Negative Control, Binding Assay, Expressing, Knockdown, Stable Transfection, Staining, Fluorescence

$O-GlcNAcylation of NONO facilitates H3K36me2 enrichment at DSBs and recruits Ku70 for NHEJ. A Left panel: O-GlcNAcylation at Ser147 stabilizes Ku70 recruitment at DNA damage sites. U2OS cells were transfected with GFP-Ku70, along with an empty vector, SFB-NONO WT, or the S147A mutant. Representative images demonstrated the dynamic recruitment of GFP-Ku70 to DNA damage sites. Data were presented as mean ± SEM. Scale bar, 10 μm. Right panel: Quantification of the time course of GFP-Ku70 recruitment following laser microirradiation. B Upper panel: O-GlcNAcylation at Ser147 stabilizes the binding of Ku70 to chromatin. HEK293T cells were transfected with an empty vector, SFB-NONO WT, or the S147A mutant and exposed to 10 Gy of IR. Following IR, cells were allowed to recover for different durations. Whole cell extract (WCE) and chromatin fractions were prepared and subjected to IP and Western blot analysis with the indicated antibodies. Lower panel: Quantification of Ku70 level, normalized to H3 levels in chromatin and presented as fold change relative to control samples ( n = 3 per condition). C Upper panel: Impact of O-GlcNAcylation at Ser147 on H3K36 demethylation levels. Stable HEK293T cells with NONO knockdown were reconstituted with shRNA-resistant NONO WT or S147A mutant. These cells were treated with IR and examined via western blot analysis with the indicated antibodies to assess H3K36me2 levels. Lower panel: Quantification of H3K36me2 level, normalized to H3 levels in chromatin and presented as fold change relative to control samples ( n = 3 per condition)$

Journal: Genome Biology

Article Title: O-GlcNAcylation of NONO mediates alternative splicing of SETMAR and facilitates NHEJ repair

doi: 10.1186/s13059-026-03930-5

Figure Lengend Snippet: O-GlcNAcylation of NONO facilitates H3K36me2 enrichment at DSBs and recruits Ku70 for NHEJ. A Left panel: O-GlcNAcylation at Ser147 stabilizes Ku70 recruitment at DNA damage sites. U2OS cells were transfected with GFP-Ku70, along with an empty vector, SFB-NONO WT, or the S147A mutant. Representative images demonstrated the dynamic recruitment of GFP-Ku70 to DNA damage sites. Data were presented as mean ± SEM. Scale bar, 10 μm. Right panel: Quantification of the time course of GFP-Ku70 recruitment following laser microirradiation. B Upper panel: O-GlcNAcylation at Ser147 stabilizes the binding of Ku70 to chromatin. HEK293T cells were transfected with an empty vector, SFB-NONO WT, or the S147A mutant and exposed to 10 Gy of IR. Following IR, cells were allowed to recover for different durations. Whole cell extract (WCE) and chromatin fractions were prepared and subjected to IP and Western blot analysis with the indicated antibodies. Lower panel: Quantification of Ku70 level, normalized to H3 levels in chromatin and presented as fold change relative to control samples ( n = 3 per condition). C Upper panel: Impact of O-GlcNAcylation at Ser147 on H3K36 demethylation levels. Stable HEK293T cells with NONO knockdown were reconstituted with shRNA-resistant NONO WT or S147A mutant. These cells were treated with IR and examined via western blot analysis with the indicated antibodies to assess H3K36me2 levels. Lower panel: Quantification of H3K36me2 level, normalized to H3 levels in chromatin and presented as fold change relative to control samples ( n = 3 per condition)

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Binding Assay, Western Blot, Control, Knockdown, shRNA

O-GlcNAcylation of NONO promotes radioresistance in hepatocellular carcinoma. A Analysis of NONO O-GlcNAcylation was conducted in human liver epithelial cells (THLE2) and hepatocellular carcinoma cells (HepG2, HCCLM9, Huh7) usingIP/Western blotting with indicated antibodies. B Generation of stable HCCLM9 cells with NONO knockdown. Western blot analysis was performed to assess the efficiency of NONO knockdown using two shRNA constructs(shNONO #1 and shNONO #2). GAPDH was used as a loading control. C Upper panel: O-GlcNAcylation at Ser147 promotes cell survival post-IR treatment. NONO-knockdown HCCLM9 cells were transfected with SFB-NONO WT or S147A mutant and subjected to clonogenic survival assays post-IR treatment. Cells were treated with the indicated doses of IR and further incubated for 7–10 days. Lower panel: Quantitative analysis of clonogenic survival assays. D Schematic diagram illustrating the radiotherapy process for NTG mice. Mice injected with control or NONO knockdown cells, as well as cells reconstituted with NONO WT or S147A and wereexposed to 8 Gy of IR twice. E O-GlcNAcylation of NONO enhances radioresistance. Mice were subcutaneously injected with 7 × 10 6 control or NONO knockdown cells, or cells reconstituted with NONO WT or S147A, and exposed to 8 Gy of IR twice or not, when tumors reached a similar size (about 100 mm. 3 ). Representative images of xenograft tumors are shown ( n = 6/group). F Quantification analysis of xenograft tumor volumes from ( E ). Data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA. ** P < 0.01, *** P < 0.001. G Hematoxylin and eosin (H&E) staining and IHC analysis of NONO and H3K36me2 in xenograft tumors were performed, comparing controland NONO knockdown groups, as well as tumors reconstituted with NONO WT or S147A ( n = 6). Scale bar, 30 μm. H Quantification of H3K36me2 levels from ( G ). Data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA. ** P < 0.01, *** P < 0.001

Journal: Genome Biology

Article Title: O-GlcNAcylation of NONO mediates alternative splicing of SETMAR and facilitates NHEJ repair

doi: 10.1186/s13059-026-03930-5

Figure Lengend Snippet: O-GlcNAcylation of NONO promotes radioresistance in hepatocellular carcinoma. A Analysis of NONO O-GlcNAcylation was conducted in human liver epithelial cells (THLE2) and hepatocellular carcinoma cells (HepG2, HCCLM9, Huh7) usingIP/Western blotting with indicated antibodies. B Generation of stable HCCLM9 cells with NONO knockdown. Western blot analysis was performed to assess the efficiency of NONO knockdown using two shRNA constructs(shNONO #1 and shNONO #2). GAPDH was used as a loading control. C Upper panel: O-GlcNAcylation at Ser147 promotes cell survival post-IR treatment. NONO-knockdown HCCLM9 cells were transfected with SFB-NONO WT or S147A mutant and subjected to clonogenic survival assays post-IR treatment. Cells were treated with the indicated doses of IR and further incubated for 7–10 days. Lower panel: Quantitative analysis of clonogenic survival assays. D Schematic diagram illustrating the radiotherapy process for NTG mice. Mice injected with control or NONO knockdown cells, as well as cells reconstituted with NONO WT or S147A and wereexposed to 8 Gy of IR twice. E O-GlcNAcylation of NONO enhances radioresistance. Mice were subcutaneously injected with 7 × 10 6 control or NONO knockdown cells, or cells reconstituted with NONO WT or S147A, and exposed to 8 Gy of IR twice or not, when tumors reached a similar size (about 100 mm. 3 ). Representative images of xenograft tumors are shown ( n = 6/group). F Quantification analysis of xenograft tumor volumes from ( E ). Data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA. ** P < 0.01, *** P < 0.001. G Hematoxylin and eosin (H&E) staining and IHC analysis of NONO and H3K36me2 in xenograft tumors were performed, comparing controland NONO knockdown groups, as well as tumors reconstituted with NONO WT or S147A ( n = 6). Scale bar, 30 μm. H Quantification of H3K36me2 levels from ( G ). Data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA. ** P < 0.01, *** P < 0.001

Techniques: Western Blot, Knockdown, shRNA, Construct, Control, Transfection, Mutagenesis, Incubation, Injection, Staining

Proposed working model of NONO O-GlcNAcylation regulating NHEJ-mediated DNA damage repair

Journal: Genome Biology

Article Title: O-GlcNAcylation of NONO mediates alternative splicing of SETMAR and facilitates NHEJ repair

doi: 10.1186/s13059-026-03930-5

Figure Lengend Snippet: Proposed working model of NONO O-GlcNAcylation regulating NHEJ-mediated DNA damage repair

Techniques:

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1) Product Images from "Essential role of NONO-HOXA1-Wnt axis in cardiomyocyte differentiation"

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