npm1 aid gfp puror addgene  (Addgene inc)

Journal: Molecular cell

Article Title: The nucleolar granular component mediates genome-nucleolus interactions and establishes their repressive chromatin states.

doi: 10.1016/j.molcel.2025.05.004

Figure Lengend Snippet: Figure 2. NPM1 associates with NADs (A) Integrative genomics viewer (IGV) track displaying NPM1 genome occupancy and NAD profiles from nucleolar-DamID11 of chromosome 5 in mESCs. (B) Genomic annotations of NPM1-bound regions at NAD and non-NADs of mESCs. Promoter corresponds to ±3 kb regions from transcription start sites. (C) NPM1 distribution on NADs and LAD-only sequences in mESCs. (D) Volcano plot showing fold change (log2 values) in transcript levels of mESCs + siRNA-Npm1 vs. mESC + siRNA-control. Gene expression values of three replicates were averaged and selected for 1.5-fold changes and p < 0.05. (E) Top 10 Gene Ontology (GO) terms as determined using database for annotation, visualization, and integrated discovery (DAVID) for genes upregulated and downregulated upon NPM1-KD. (F) Pie chart showing the number of genes located at NAD-only and NAD/LAD regions that are significantly upregulated or downregulated in mESC upon NPM1-KD.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Experimental models: Cell lines One hundred and twenty-nine mouse embryonic stem cells (E14 line) Savi�c et al.40 N/A HEK293T ATCC N/A NIH3T3 ATCC N/A HP1α/β-cDKO mESC Ballmer et al.24 N/A hsp-TetO-H2B-Dam-NoLS-DD mESCs Bersaglieri et al.11 N/A FLAG-HA-NPM1 mESCs This work N/A FKBP-NPM1 mESCs This work N/A Oligonucleotides Oligonucleotides for qPCR and siRNA This work Table S7 Recombinant DNA Super PiggyBac Transposase Expression Vector SBI System Biosciences PB210PA-1 pSpCas9(BB)-2A-GFP Addgene 48138 pX330 Mouse 5’ Npm1 gRNA Addgene 127900 5’ Npm1-AID-GFP-PuroR Addgene 127899 5’ Npm1-FLAG-HA-PuroR This work N/A 5’ Npm1- FKBP-PuroR This work N/A CMV-H2B-GFP Bersaglieri et al.11 N/A CMV-H2B-GFP-NoLS Bersaglieri et al.11 N/A shRNA-Control This work N/A shRNA-Npm1 This work N/A CMV-HA-G9a This work N/A EF1α-NPM1 This work N/A EF1α-NPM1ΔC This work N/A EF1α-NPM1ΔDBD This work N/A Software and algorithms SortMeRNA version 2.1 Kopylova et al.41 https://github.com/sortmerna/sortmerna Trimmomatic version 0.40 Bolger et al.42 http://www.usadellab.org/cms/?page=trimmomatic deepTools 3.5.0 Ramı́rez et al.43 https://deeptools.readthedocs.io/en/3.5.0/ content/installation.html Integrative Genome Viewer, version 2.5.2 Robinson et al.44 https://igv.org bedtools 2.24.0 Quinlan and Hall45 https://launchpad.net/ubuntu/+source/ bedtools/2.24.0-1 R 3.4.3 R-project https://cran-archive.r-project.org/bin/ windows/base/old/3.4.3/ Fiji 2.9.0/1.53t Schneider et al.46 https://imagej.net/software/fiji/ Prism GraphPad 10.1.0 GraphPad Software https://www.graphpad.com/features Other Lipofectamine® RNAiMAX Transfection Reagent Invitrogen 13778150 LipofectamineTM Stem Transfection reagent Thermo Fischer Scientific STEM00001 Transit-X2 transfection reagent Mirus MIR 6004 ll OPEN ACCESSArticle Molecular Cell 85, 1–11.e1–e6, June 5, 2025 e2 (Corning® CellBIND® surface) coated with 0.1% gelatin (Sigma, G1890) without a feeder layer.

Techniques: Control, Gene Expression

Journal: Molecular cell

Article Title: The nucleolar granular component mediates genome-nucleolus interactions and establishes their repressive chromatin states.

doi: 10.1016/j.molcel.2025.05.004

Figure Lengend Snippet: Figure 3. NPM1 regulates G9a-mediated H3K9me2 at NADs (A) Representative immunofluorescence images showing H3K9me2 distribution in mESCs treated with siRNA-control or siRNA-Npm1. The magnification on the right shows the distribution of H3K9me2 around nucleoli, forming a ring-like shape that is destroyed in the absence of NPM1. Upstream binding transcription factor (UBF) serves as a nucleolar marker. Scale bar represents 5 μm. The corresponding DAPI staining is shown in Figure S3F. (B) Quantification of cells showing the perinucleolar H3K9me2 ring in mESCs upon depletion of G9a, SUV39H1/2, or SETDB1. Data are from three independent experiments. Error bars represent SD. Statistical significance (p values) was calculated using the Mann-Whitney test (***p < 0.001).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Experimental models: Cell lines One hundred and twenty-nine mouse embryonic stem cells (E14 line) Savi�c et al.40 N/A HEK293T ATCC N/A NIH3T3 ATCC N/A HP1α/β-cDKO mESC Ballmer et al.24 N/A hsp-TetO-H2B-Dam-NoLS-DD mESCs Bersaglieri et al.11 N/A FLAG-HA-NPM1 mESCs This work N/A FKBP-NPM1 mESCs This work N/A Oligonucleotides Oligonucleotides for qPCR and siRNA This work Table S7 Recombinant DNA Super PiggyBac Transposase Expression Vector SBI System Biosciences PB210PA-1 pSpCas9(BB)-2A-GFP Addgene 48138 pX330 Mouse 5’ Npm1 gRNA Addgene 127900 5’ Npm1-AID-GFP-PuroR Addgene 127899 5’ Npm1-FLAG-HA-PuroR This work N/A 5’ Npm1- FKBP-PuroR This work N/A CMV-H2B-GFP Bersaglieri et al.11 N/A CMV-H2B-GFP-NoLS Bersaglieri et al.11 N/A shRNA-Control This work N/A shRNA-Npm1 This work N/A CMV-HA-G9a This work N/A EF1α-NPM1 This work N/A EF1α-NPM1ΔC This work N/A EF1α-NPM1ΔDBD This work N/A Software and algorithms SortMeRNA version 2.1 Kopylova et al.41 https://github.com/sortmerna/sortmerna Trimmomatic version 0.40 Bolger et al.42 http://www.usadellab.org/cms/?page=trimmomatic deepTools 3.5.0 Ramı́rez et al.43 https://deeptools.readthedocs.io/en/3.5.0/ content/installation.html Integrative Genome Viewer, version 2.5.2 Robinson et al.44 https://igv.org bedtools 2.24.0 Quinlan and Hall45 https://launchpad.net/ubuntu/+source/ bedtools/2.24.0-1 R 3.4.3 R-project https://cran-archive.r-project.org/bin/ windows/base/old/3.4.3/ Fiji 2.9.0/1.53t Schneider et al.46 https://imagej.net/software/fiji/ Prism GraphPad 10.1.0 GraphPad Software https://www.graphpad.com/features Other Lipofectamine® RNAiMAX Transfection Reagent Invitrogen 13778150 LipofectamineTM Stem Transfection reagent Thermo Fischer Scientific STEM00001 Transit-X2 transfection reagent Mirus MIR 6004 ll OPEN ACCESSArticle Molecular Cell 85, 1–11.e1–e6, June 5, 2025 e2 (Corning® CellBIND® surface) coated with 0.1% gelatin (Sigma, G1890) without a feeder layer.

Techniques: Immunofluorescence, Control, Binding Assay, Marker, Staining, MANN-WHITNEY

Journal: Molecular cell

Article Title: The nucleolar granular component mediates genome-nucleolus interactions and establishes their repressive chromatin states.

doi: 10.1016/j.molcel.2025.05.004

Figure Lengend Snippet: Figure 2. NPM1 associates with NADs (A) Integrative genomics viewer (IGV) track displaying NPM1 genome occupancy and NAD profiles from nucleolar-DamID11 of chromosome 5 in mESCs. (B) Genomic annotations of NPM1-bound regions at NAD and non-NADs of mESCs. Promoter corresponds to ±3 kb regions from transcription start sites. (C) NPM1 distribution on NADs and LAD-only sequences in mESCs. (D) Volcano plot showing fold change (log2 values) in transcript levels of mESCs + siRNA-Npm1 vs. mESC + siRNA-control. Gene expression values of three replicates were averaged and selected for 1.5-fold changes and p < 0.05. (E) Top 10 Gene Ontology (GO) terms as determined using database for annotation, visualization, and integrated discovery (DAVID) for genes upregulated and downregulated upon NPM1-KD. (F) Pie chart showing the number of genes located at NAD-only and NAD/LAD regions that are significantly upregulated or downregulated in mESC upon NPM1-KD.

Article Snippet: To generate FLAG-HA tagged and FKBP-tagged NPM1 mESC lines, cells were co-transfected with a plasmid expressing Cas9 protein and the gRNA sequence targeting the NPM1 locus (pX330 Mouse 5’ Npm1 gRNA, a gift from Mark Groudine, Addgene plasmid #127900; http://n2t.net/addgene:127900; RRID:Addgene_127900) and the HDR donor plasmid derived from mouse 5’ Npm1-AID-GFP-PuroR plasmid (a gift from Mark Groudine, Addgene plasmid #127899;http://n2t.net/addgene:127899; RRID:Addgene_127899) in which AID-GFP sequences were replaced with FLAG-HA or FKBP sequence (synthesized from IDT).

Techniques: Control, Gene Expression

Journal: Molecular cell

Article Title: The nucleolar granular component mediates genome-nucleolus interactions and establishes their repressive chromatin states.

doi: 10.1016/j.molcel.2025.05.004

Figure Lengend Snippet: Figure 3. NPM1 regulates G9a-mediated H3K9me2 at NADs (A) Representative immunofluorescence images showing H3K9me2 distribution in mESCs treated with siRNA-control or siRNA-Npm1. The magnification on the right shows the distribution of H3K9me2 around nucleoli, forming a ring-like shape that is destroyed in the absence of NPM1. Upstream binding transcription factor (UBF) serves as a nucleolar marker. Scale bar represents 5 μm. The corresponding DAPI staining is shown in Figure S3F. (B) Quantification of cells showing the perinucleolar H3K9me2 ring in mESCs upon depletion of G9a, SUV39H1/2, or SETDB1. Data are from three independent experiments. Error bars represent SD. Statistical significance (p values) was calculated using the Mann-Whitney test (***p < 0.001).

Article Snippet: To generate FLAG-HA tagged and FKBP-tagged NPM1 mESC lines, cells were co-transfected with a plasmid expressing Cas9 protein and the gRNA sequence targeting the NPM1 locus (pX330 Mouse 5’ Npm1 gRNA, a gift from Mark Groudine, Addgene plasmid #127900; http://n2t.net/addgene:127900; RRID:Addgene_127900) and the HDR donor plasmid derived from mouse 5’ Npm1-AID-GFP-PuroR plasmid (a gift from Mark Groudine, Addgene plasmid #127899;http://n2t.net/addgene:127899; RRID:Addgene_127899) in which AID-GFP sequences were replaced with FLAG-HA or FKBP sequence (synthesized from IDT).

Techniques: Immunofluorescence, Control, Binding Assay, Marker, Staining, MANN-WHITNEY