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crispr/cas9 knockdown kit targeting human cox-2  (Santa Cruz Biotechnology)


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    Santa Cruz Biotechnology crispr/cas9 knockdown kit targeting human cox-2
    Crispr/Cas9 Knockdown Kit Targeting Human Cox 2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/crispr/cas9 knockdown kit targeting human cox-2/product/Santa Cruz Biotechnology
    Average 90 stars, based on 1 article reviews
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    (A) Schematic of <t>CRISPR/Cas9</t> strategy targeting exons 3–5 of <t>NMNAT1</t> in U-2OS cells to generate a frameshift mutation and truncated protein. (B) Western blot confirming NMNAT1 depletion in NMNAT1 KO cells. Quantification normalized to H3 is shown (mean ± SEM, n = 3; p < 0.01, unpaired t-test). (C) Total NAD⁺ levels are significantly reduced in NMNAT1 KO cells as measured by NAD⁺/NADH assay (mean ± SEM, n = 3; p < 0.01, unpaired t-test). (D) ATP levels are significantly decreased in NMNAT1 KO cells (mean ± SEM, n = 5; p < 0.01, unpaired t-test). (E) Calcein-AM viability assay reveals no significant change in NMNAT1 KO cell viability (mean ± SEM, n = 3). (F) Growth curve analysis shows comparable proliferation rates between NMNAT1 KO and control cells over five days (mean ± SEM, n = 2). (G) Model of NMNAT1-regulated transcription via NAD⁺-dependent PARP1 activation and promoter-associated PARylation. Adapted from Zhang et al., J. Biol. Chem. (2009, 2012). (H) PARP1 protein levels remain unchanged in NMNAT1 KO cells. Quantification shown (mean ± SEM, n = 1; p < 0.01, unpaired t-test). (I) Global PAR levels are significantly reduced in NMNAT1 KO cells, indicating loss of PARP1 activity (mean ± SEM, n = 1; p < 0.01, unpaired t-test).
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    Image Search Results


    (A) Schematic of CRISPR/Cas9 strategy targeting exons 3–5 of NMNAT1 in U-2OS cells to generate a frameshift mutation and truncated protein. (B) Western blot confirming NMNAT1 depletion in NMNAT1 KO cells. Quantification normalized to H3 is shown (mean ± SEM, n = 3; p < 0.01, unpaired t-test). (C) Total NAD⁺ levels are significantly reduced in NMNAT1 KO cells as measured by NAD⁺/NADH assay (mean ± SEM, n = 3; p < 0.01, unpaired t-test). (D) ATP levels are significantly decreased in NMNAT1 KO cells (mean ± SEM, n = 5; p < 0.01, unpaired t-test). (E) Calcein-AM viability assay reveals no significant change in NMNAT1 KO cell viability (mean ± SEM, n = 3). (F) Growth curve analysis shows comparable proliferation rates between NMNAT1 KO and control cells over five days (mean ± SEM, n = 2). (G) Model of NMNAT1-regulated transcription via NAD⁺-dependent PARP1 activation and promoter-associated PARylation. Adapted from Zhang et al., J. Biol. Chem. (2009, 2012). (H) PARP1 protein levels remain unchanged in NMNAT1 KO cells. Quantification shown (mean ± SEM, n = 1; p < 0.01, unpaired t-test). (I) Global PAR levels are significantly reduced in NMNAT1 KO cells, indicating loss of PARP1 activity (mean ± SEM, n = 1; p < 0.01, unpaired t-test).

    Journal: bioRxiv

    Article Title: NMNAT1 Binding at Promoters and Enhancers Couples NAD + Synthesis to RNA Polymerase II Engagement

    doi: 10.1101/2025.04.30.651499

    Figure Lengend Snippet: (A) Schematic of CRISPR/Cas9 strategy targeting exons 3–5 of NMNAT1 in U-2OS cells to generate a frameshift mutation and truncated protein. (B) Western blot confirming NMNAT1 depletion in NMNAT1 KO cells. Quantification normalized to H3 is shown (mean ± SEM, n = 3; p < 0.01, unpaired t-test). (C) Total NAD⁺ levels are significantly reduced in NMNAT1 KO cells as measured by NAD⁺/NADH assay (mean ± SEM, n = 3; p < 0.01, unpaired t-test). (D) ATP levels are significantly decreased in NMNAT1 KO cells (mean ± SEM, n = 5; p < 0.01, unpaired t-test). (E) Calcein-AM viability assay reveals no significant change in NMNAT1 KO cell viability (mean ± SEM, n = 3). (F) Growth curve analysis shows comparable proliferation rates between NMNAT1 KO and control cells over five days (mean ± SEM, n = 2). (G) Model of NMNAT1-regulated transcription via NAD⁺-dependent PARP1 activation and promoter-associated PARylation. Adapted from Zhang et al., J. Biol. Chem. (2009, 2012). (H) PARP1 protein levels remain unchanged in NMNAT1 KO cells. Quantification shown (mean ± SEM, n = 1; p < 0.01, unpaired t-test). (I) Global PAR levels are significantly reduced in NMNAT1 KO cells, indicating loss of PARP1 activity (mean ± SEM, n = 1; p < 0.01, unpaired t-test).

    Article Snippet: An NMNAT1 knockout cell line was generated using a commercially available NMNAT1 CRISPR/Cas9 knockout plasmid kit (sc-403085, Santa Cruz Biotechnology, Inc.) and an NMNAT1 HDR plasmid (sc-403085-HDR, Santa Cruz Biotechnology, Inc.).

    Techniques: CRISPR, Mutagenesis, Western Blot, Viability Assay, Control, Activation Assay, Activity Assay

    (A) Schematic of the CUT&Tag workflow to profile NMNAT1 chromatin binding in U-2OS cells. The signal from NMNAT1 KO cells was subtracted to define high-confidence peaks. (B) Motif analysis of NMNAT1-bound regions performed using HOMER shows enrichment of AP-1 transcription factor motifs, including Fra1/2, Fos, JunB, and Atf3. (C) Genomic distribution of 16,751 NMNAT1 peaks identified from CUT&Tag analysis. Peaks were annotated relative to gene features, including promoters (27.7%), introns (32.8%), and distal intergenic regions (29%). (D) Metaplot and heatmap of NMNAT1 signal centered on transcription start sites (TSS ±1 kb), showing non-uniform enrichment across promoter regions. (E) Metaplot of NMNAT1 signal centered on known U-2OS enhancers from EnhancerAtlas2.0. (F) Distribution (percentages) of NMNAT1-bound at known enhancers (U-2OS EnhancerAtlas2.0).

    Journal: bioRxiv

    Article Title: NMNAT1 Binding at Promoters and Enhancers Couples NAD + Synthesis to RNA Polymerase II Engagement

    doi: 10.1101/2025.04.30.651499

    Figure Lengend Snippet: (A) Schematic of the CUT&Tag workflow to profile NMNAT1 chromatin binding in U-2OS cells. The signal from NMNAT1 KO cells was subtracted to define high-confidence peaks. (B) Motif analysis of NMNAT1-bound regions performed using HOMER shows enrichment of AP-1 transcription factor motifs, including Fra1/2, Fos, JunB, and Atf3. (C) Genomic distribution of 16,751 NMNAT1 peaks identified from CUT&Tag analysis. Peaks were annotated relative to gene features, including promoters (27.7%), introns (32.8%), and distal intergenic regions (29%). (D) Metaplot and heatmap of NMNAT1 signal centered on transcription start sites (TSS ±1 kb), showing non-uniform enrichment across promoter regions. (E) Metaplot of NMNAT1 signal centered on known U-2OS enhancers from EnhancerAtlas2.0. (F) Distribution (percentages) of NMNAT1-bound at known enhancers (U-2OS EnhancerAtlas2.0).

    Article Snippet: An NMNAT1 knockout cell line was generated using a commercially available NMNAT1 CRISPR/Cas9 knockout plasmid kit (sc-403085, Santa Cruz Biotechnology, Inc.) and an NMNAT1 HDR plasmid (sc-403085-HDR, Santa Cruz Biotechnology, Inc.).

    Techniques: Binding Assay

    (A) Schematic of the integrative analysis pipeline combining NMNAT1 CUT&Tag with RNA-seq data to assess the relationship between chromatin occupancy and gene expression in U-2OS cells. (B) Metaplot of NMNAT1 signal intensity at ±1 kb of the transcription start site (TSS) and transcription end site (TES) across unchanged (black), upregulated (red), and downregulated (blue) genes. NMNAT1 shows the highest enrichment at the TSS of downregulated genes. (C) Log₂ odds ratio plot quantifying the enrichment of NMNAT1 binding at the promoter-TSS of downregulated genes (blue) compared to unchanged and upregulated genes (red). Significant enrichment of NMNAT1 peaks was observed at downregulated genes ( p = 1.309e −24 ) but not at upregulated genes (See also Figure S1 for complete statistical analysis). (D) Log₂ odds ratio plot quantifying the enrichment of NMNAT1 binding at enhancers of downregulated genes (blue) compared to unchanged and upregulated genes (red). Significant enrichment of NMNAT1-enhancer bound was observed at downregulated genes ( p = 3.786e −12 ) but not at upregulated genes (See also Figure S1 for complete statistical analysis). (E) Genome browser tracks of NMNAT1 CUT&Tag signal in U-2OS control subtracted from CUT&Tag signal in NMNAT1 KO cells at selected target gene promoters. (F) Gene Ontology (GO) analysis of NMNAT1-bound downregulated genes. Significantly enriched terms include cell cycle regulation, DNA replication, and extracellular matrix organization. GO terms are ranked by −log₁₀ adjusted p -value.

    Journal: bioRxiv

    Article Title: NMNAT1 Binding at Promoters and Enhancers Couples NAD + Synthesis to RNA Polymerase II Engagement

    doi: 10.1101/2025.04.30.651499

    Figure Lengend Snippet: (A) Schematic of the integrative analysis pipeline combining NMNAT1 CUT&Tag with RNA-seq data to assess the relationship between chromatin occupancy and gene expression in U-2OS cells. (B) Metaplot of NMNAT1 signal intensity at ±1 kb of the transcription start site (TSS) and transcription end site (TES) across unchanged (black), upregulated (red), and downregulated (blue) genes. NMNAT1 shows the highest enrichment at the TSS of downregulated genes. (C) Log₂ odds ratio plot quantifying the enrichment of NMNAT1 binding at the promoter-TSS of downregulated genes (blue) compared to unchanged and upregulated genes (red). Significant enrichment of NMNAT1 peaks was observed at downregulated genes ( p = 1.309e −24 ) but not at upregulated genes (See also Figure S1 for complete statistical analysis). (D) Log₂ odds ratio plot quantifying the enrichment of NMNAT1 binding at enhancers of downregulated genes (blue) compared to unchanged and upregulated genes (red). Significant enrichment of NMNAT1-enhancer bound was observed at downregulated genes ( p = 3.786e −12 ) but not at upregulated genes (See also Figure S1 for complete statistical analysis). (E) Genome browser tracks of NMNAT1 CUT&Tag signal in U-2OS control subtracted from CUT&Tag signal in NMNAT1 KO cells at selected target gene promoters. (F) Gene Ontology (GO) analysis of NMNAT1-bound downregulated genes. Significantly enriched terms include cell cycle regulation, DNA replication, and extracellular matrix organization. GO terms are ranked by −log₁₀ adjusted p -value.

    Article Snippet: An NMNAT1 knockout cell line was generated using a commercially available NMNAT1 CRISPR/Cas9 knockout plasmid kit (sc-403085, Santa Cruz Biotechnology, Inc.) and an NMNAT1 HDR plasmid (sc-403085-HDR, Santa Cruz Biotechnology, Inc.).

    Techniques: RNA Sequencing, Gene Expression, Binding Assay, Control

    (A) Schematic model of NMNAT1 function in transcriptional regulation. NMNAT1 is proposed to support RNA-Pol II pausing and release via NAD⁺-dependent PARP1 activity and NELF, adding to the previous model . (B) Schematic of CUT&Tag experimental workflow used to profile RNA-Pol II Ser5P occupancy in U-2OS control and NMNAT1 KO cells. (C) Differential peak analysis of 35,373 RNA-Pol II Ser5P peaks identifies 1,748 (5%) with decreased and 2,132 (6%) with increased occupancy in NMNAT1 KO cells relative to controls. Analysis performed using the GoodpeaksScript pipeline. (D) Metaplot of RNA-Pol II Ser5P signal across downregulated (left panel), upregulated (middle panel), and unchanged (right panel) DEGs. Reduced signal is observed specifically at downregulated genes in NMNAT1 KO cells. (E) Log₂ odds ratio plot quantifying the enrichment of RNA Polymerase II (Pol II) occupancy changes at downregulated (blue), unchanged, and upregulated (red) genes. Significant enrichment of RNA-Pol II-decreased peaks was observed at downregulated genes (p = 2.90e −84 ) but not at upregulated genes. Conversely, RNA-Pol II–increased peaks were significantly enriched at upregulated genes (p = 2.25e −27 ) but not at downregulated genes (see also Figure S2 for complete statistical analysis). (F) Genome browser tracks RNA-Pol II Ser5P occupancy at representative target genes in control and NMNAT1 KO U-2OS cells.

    Journal: bioRxiv

    Article Title: NMNAT1 Binding at Promoters and Enhancers Couples NAD + Synthesis to RNA Polymerase II Engagement

    doi: 10.1101/2025.04.30.651499

    Figure Lengend Snippet: (A) Schematic model of NMNAT1 function in transcriptional regulation. NMNAT1 is proposed to support RNA-Pol II pausing and release via NAD⁺-dependent PARP1 activity and NELF, adding to the previous model . (B) Schematic of CUT&Tag experimental workflow used to profile RNA-Pol II Ser5P occupancy in U-2OS control and NMNAT1 KO cells. (C) Differential peak analysis of 35,373 RNA-Pol II Ser5P peaks identifies 1,748 (5%) with decreased and 2,132 (6%) with increased occupancy in NMNAT1 KO cells relative to controls. Analysis performed using the GoodpeaksScript pipeline. (D) Metaplot of RNA-Pol II Ser5P signal across downregulated (left panel), upregulated (middle panel), and unchanged (right panel) DEGs. Reduced signal is observed specifically at downregulated genes in NMNAT1 KO cells. (E) Log₂ odds ratio plot quantifying the enrichment of RNA Polymerase II (Pol II) occupancy changes at downregulated (blue), unchanged, and upregulated (red) genes. Significant enrichment of RNA-Pol II-decreased peaks was observed at downregulated genes (p = 2.90e −84 ) but not at upregulated genes. Conversely, RNA-Pol II–increased peaks were significantly enriched at upregulated genes (p = 2.25e −27 ) but not at downregulated genes (see also Figure S2 for complete statistical analysis). (F) Genome browser tracks RNA-Pol II Ser5P occupancy at representative target genes in control and NMNAT1 KO U-2OS cells.

    Article Snippet: An NMNAT1 knockout cell line was generated using a commercially available NMNAT1 CRISPR/Cas9 knockout plasmid kit (sc-403085, Santa Cruz Biotechnology, Inc.) and an NMNAT1 HDR plasmid (sc-403085-HDR, Santa Cruz Biotechnology, Inc.).

    Techniques: Activity Assay, Control

    Model: Depletion of NMNAT1 disrupts local nuclear NAD⁺ synthesis, impairing PARP1 activity and leading to poor engagement of RNA-Pol II at gene promoters. Previous models proposed that PARP1 facilitates promoter-proximal pause release of RNA-Pol II via PARylation of NELF; however, our data reveal that NMNAT1 KO cells exhibit reduced RNA-Pol II occupancy at transcription start sites of downregulated genes, indicating a defect in RNA-Pol II recruitment or stabilization. This suggests that NMNAT1-derived NAD⁺ may enable PARP1 to modify additional chromatin-associated factors required for RNA-Pol II engagement.

    Journal: bioRxiv

    Article Title: NMNAT1 Binding at Promoters and Enhancers Couples NAD + Synthesis to RNA Polymerase II Engagement

    doi: 10.1101/2025.04.30.651499

    Figure Lengend Snippet: Model: Depletion of NMNAT1 disrupts local nuclear NAD⁺ synthesis, impairing PARP1 activity and leading to poor engagement of RNA-Pol II at gene promoters. Previous models proposed that PARP1 facilitates promoter-proximal pause release of RNA-Pol II via PARylation of NELF; however, our data reveal that NMNAT1 KO cells exhibit reduced RNA-Pol II occupancy at transcription start sites of downregulated genes, indicating a defect in RNA-Pol II recruitment or stabilization. This suggests that NMNAT1-derived NAD⁺ may enable PARP1 to modify additional chromatin-associated factors required for RNA-Pol II engagement.

    Article Snippet: An NMNAT1 knockout cell line was generated using a commercially available NMNAT1 CRISPR/Cas9 knockout plasmid kit (sc-403085, Santa Cruz Biotechnology, Inc.) and an NMNAT1 HDR plasmid (sc-403085-HDR, Santa Cruz Biotechnology, Inc.).

    Techniques: Activity Assay, Derivative Assay