self targeting gfp construct  (Addgene inc)

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Heatmap of log 2 FC values for ubiquitylated peptides in MDS-L FBXO11-KO versus WT cells. Shown are hits with a FC of greater than |0.5|. Known RNA-binding proteins are labeled in red. Data were reanalyzed from ref. . ( B ) Scatter plot of the results from co-IP and MS identification of endogenous FBXO11 complexes in F-36P cell nuclear fractions. Shown are the –log( P value) against the FC enrichment of individual proteins identified in the FBXO11 IP versus the IgG control IP. The blue box highlights proteins with a P value of less than 0.05 and a FC of greater than 1.5. The P value was derived by G test. ( C ) STRING network analysis of FBXO11-interacting proteins identified by MS. Only input nodes are shown, with the threshold cutoff of medium confidence at 0.7. The thickness of the lines connecting nodes indicates the relative strength of evidence supporting the protein-protein interactions. ( D ) Strategy for the FBXO11 substrate-focused CRISPR/Cas9 screen, in which the gRNA library encompasses all of the differentially ubiquitylated peptides identified from the ubiquitin proteomics experiment represented in A . ( E ) Summary table of significant hits from the MAGeCK (Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout) analysis of the CRISPR screen in D , demonstrating selective enrichment or depletion of guides in sgCTRL or sgFBXO11 colonies. ( F ) Schematic overview of the integrated multiomics approach to identify relevant candidate FBXO11 substrates meeting the listed criteria, revealing NPM1 and HNRNPU. ( G ) FC in individual gRNA read counts for NPM1 and HNRNPU in the colony-forming assay (day 10) versus initial representation (day 0) in the CRISPR screen. The FC for individual guides was compared between sgCTRL and sgFBXO11 experimental groups, shown on the graphs. n = 6 guides per gene, in 2 independent biological replicates of the CRISPR screen. * P < 0.05, by 2-tailed, paired t test.

Article Snippet: Human lentiviral shRNA constructs targeting FBXO11 in the pLVRU6MP-mCherry vectors were purchased from Genecopoeia.

Techniques: RNA Binding Assay, Labeling, Co-Immunoprecipitation Assay, Control, Derivative Assay, Protein-Protein interactions, CRISPR, Ubiquitin Proteomics, Genome Wide, Knock-Out

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Immunoblots of overexpressed FLAG-FBXO11 isoforms in HEK293T cells. Blotting was done for endogenous NPM1 and H2A. Endogenous NPM1 IPs were probed for FBXO11 and NPM1. ( B ) Immunoblots of co-IP FLAG-FBXO11 complexes of FBXO11 and NPM1. ( C ) Left: Input immunoblots for FLAG-FBXO11, NPM1, ubiquitin-GFP, and H2A in HEK293T cells. IPs of NPM1 were performed to detect ubiquitylation by FBXO11. Right: NPM1 versus IgG control IP, immunoblotted for GFP-ubiquitin. n = 3. ( D ) Densitometry for NPM1 poly-ubiquitin bands from C . The area quantified is 75 kDa and above. n = 3. * Q < 0.05, by Kruskal-Wallis ANOVA corrected for multiple comparisons by the Benjamini method. ( E ) Schematic depicting K248-Ub of NPM1 in its C-terminal core. The ubiquitin (Ub) footprint was identified by MS, and . ( F ) HEK239T cells were transfected with NPM1-GFP or NPM1-K248R-GFP fusions. Red outlines indicate GFP-bright regions annotated in QuPath. Scale bars: 10 μm. ( G and H ) Annotated regions quantified for circularity ( G ) and aspect ratio ( H ). n = 2. Dots represent individual NPM1 + regions.* P < 0.05 and ** P < 0.01, by 2-tailed Mann-Whitney U test. ( I ) Confocal images of human CD34 + cells expressing sh CTRL or sh FBXO11 mCherry constructs. Images are pseudocolored for FBXO11 (magenta), NPM1 (green), and DAPI (blue). Scale bars: 5 μm. ( J ) NPM1-bright objects per cell. * P < 0.05, by 2-tailed Mann-Whitney U test. ( K ) Circularity values for NPM1-bright objects in the shCTRL and shFBXO11 images. * P < 0.05, by 2-tailed Mann-Whitney U test. ( L ) Aspect ratio values for NPM1-bright objects in the shCTRL and shFBXO11 images. * P < 0.05, by 2-tailed Mann-Whitney U test. ( M ) Relative MFI values for FBXO11 signal and NPM1 signal on a per-cell basis. **** P < 0.0001, by 2-tailed t test. ( N ) Human CD34 + cells stained for FBXO11 (green) and NPM1 (magenta). Colocalization (white arrowheads) where green and magenta overlap. Scale bars: 5 μm. ( O ) Pearson correlation values for NPM1 and FBXO11 signals in the nucleolus (NPM1-bright) and nucleoplasm (NPM1-dim). Each dot represents 1 cell, with greater than 100 cells at ×63. **** P < 0.0001, by 2-tailed Mann-Whitney U test.

Article Snippet: Human lentiviral shRNA constructs targeting FBXO11 in the pLVRU6MP-mCherry vectors were purchased from Genecopoeia.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Control, Transfection, MANN-WHITNEY, Expressing, Construct, Staining

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Schematic of splicing reporter delivered to F-36P cells. ( B ) Immunoblot for FBXO11 in HEK293T lysates with an increasing transfected plasmid dose of FBXO11-long and FBXO11-short, compared with endogenous hnRNPR and SYNCRIP. ( C ) Percentage of DsRed + cells normalized to reporter-positive cells in B measured by FACS at 48 hours. * Q < 0.05 and ** Q < 0.01, by multiple 2-tailed t tests corrected for multiple comparisons. ( D ) Representative flow plots of FBXO11-long and FBXO11-short reporter-positive cells. ( E ) DsRed-eGFP + F-36P cell percentages normalized to reporter-positive cells. n = 3 independent experiments; n = 3 wells per group. ** P < 0.01, by 2-tailed t test. ( F ) FC in DsRed + cells in sgFBXO11 versus sgNT, in the context of parental F-36P cells or F-36P cells with NPM1 overexpression. ** Q < 0.01. ( G ) Representative flow plots of sgFBXO1 versus sgNT in F-36P cells overexpressing NPM1. ( H ) log 2 FC of FBXO11 expression in healthy control cells or MDS CD34 + samples, grouped by common splicing factor mutations. SF WT, splicing factor WT. * Q < 0.05, by 1-way ordinary ANOVA corrected for multiple comparisons using the Benjamini method. ( I ) Number of significant alternative splicing events determined by rMATS analysis. ( J ) Top: Schematic of junction reads versus skipped reads, used to determine exon inclusion levels for splicing events. Bottom: Exon inclusion levels for putative FBXO11-dependent splicing events in SLC22A16 and AFTPH in MDS CD34 + samples that were null for any splicing factor mutation. Each dot represents 1 patient.

Article Snippet: Human lentiviral shRNA constructs targeting FBXO11 in the pLVRU6MP-mCherry vectors were purchased from Genecopoeia.

Techniques: Western Blot, Transfection, Plasmid Preparation, Over Expression, Expressing, Control, Alternative Splicing, Mutagenesis

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Workflow to isolate CD34 + cell fractions from healthy donor ( n = 6) and MDS patient ( n = 13) samples, to normalize to 25,000 live cells per replicate tube, and perform total quantitative proteomics by DIA of peptide spectra. Illustration was created in BioRender.com. ( B ) Volcano plot of differentially detected proteins in MDS samples versus healthy controls. Each data point indicates 1 protein, with coordinates reflecting the –log 10 of the P value against its log 2 FC for patients with MDS ( n = 13) versus healthy donors ( n = 6). Color-coding indicates a P value cutoff of less than 0.05 and a log 2 FC cutoff >|0.5|. ( C ) Heatmap of the top 400 differentially expressed proteins in MDS versus healthy controls. Unsupervised hierarchical clustering of patient samples was performed using ClustVis. Each column represents 1 replicate tube from the indicated patient samples below the map. R1, run 1; R2, run 2. ( D ) GO molecular function analysis of cluster 2 proteins, the largest cluster downregulated in MDS. ( E ) GO molecular function analysis of cluster 8 proteins, the largest cluster upregulated in MDS. ( F ) Schematic depicting the data integration performed using Cytoscape to quantify the FBXO11 interactome in primary MDS HSPCs. Schematic was created in BioRender.com. ( G ) Resultant data visualization of the integrated proteomics analysis performed in F . Blue indicates down in MDS; red indicates up in MDS. ( H ) Normalized enrichment scores from GSEA of the significantly differentially expressed proteins in the MDS proteome versus healthy controls.

Article Snippet: Human lentiviral shRNA constructs targeting FBXO11 in the pLVRU6MP-mCherry vectors were purchased from Genecopoeia.

Techniques: Quantitative Proteomics

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Upstream regulator analysis of FBXO11-interacting proteins performed using IPA software. MYC was identified as a significant upstream regulator of the FBXO11 interactome ( P = 1.07 × 10 15 ). Shown is the MYC mechanistic network in hierarchical format, with FBXO11-interacting proteins in blue. ( B ) Integrative genomics viewer gene track of FBXO11 from MYC ChIP-Seq results in ENCODE. ( C and D ) FC of MYC binding to FBXO11 promoter sequences, calculated from MYC ChIP-qPCR in F-36P and MDS92 cells. TLR2 agonist (CU-T12-9) treatment resulted in a complete loss of MYC expression. * Q < 0.05, by 2-way ANOVA corrected for multiple comparisons using the Benjamini method. ( E ) TLR2 peptide intensity determined by quantitative proteomics from MDS patient sample replicates in which TLR2 peptides were detected. Each dot indicates 1 replicate. **** P < 0.0001, by 2-tailed Mann Whitney U test.

Article Snippet: Human lentiviral shRNA constructs targeting FBXO11 in the pLVRU6MP-mCherry vectors were purchased from Genecopoeia.

Techniques: Software, ChIP-sequencing, Binding Assay, ChIP-qPCR, Expressing, Quantitative Proteomics, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Immunoblots of FBXO11, NPM1, and H3 with densitometry. Each sample was normalized to H3, and then to controls. ( B ) Densitometric values of FBXO11 and NPM1 from A . Pearson correlation for all pairs of values and line of best fit with a 95% CI. ( C ) Cell fitness assay using primary CD34 + cells. FC in indel percentages at the end of the assay versus the initial read. For double knockout, the indel percentage tracks NPM1 edits. * Q < 0.05 and ** Q < 0.01, by 2-group specific longitudinal mixed-effects analysis corrected for multiple comparisons. ( D ) Number of colonies. * Q < 0.05, ** Q < 0.01, and *** Q < 0.001, by multiple t tests corrected for multiple comparisons. n = 3. ( E ) Number of colonies. n = 62 with 3 wells per assay. * Q < 0.05 and ** Q < 0.01, by t tests on pooled replicates corrected for multiple comparisons. ( F ) Growth curve of F-36P GFP + vector or FBXO11-overexpressing cells. n = 9 wells per group. **** P < 0.0001, by t test from the linear mixed-effects model with wells as a random effect. ( G ) Growth curve of MDS92 GFP + vector or FBXO11-overexpressing cells. n = 9 wells per group. **** P < 0.0001, by t test from the linear mixed-effects model with wells as a random effect. ( H ) Schematic of the RUNX1-driven MDS mouse model on an inducible Mx1-Cre + Fbxo11 +/+ or Fbxo11 +/– background. ( I ) Percentage of GFP + peripheral blood cells isolated from Fbxo11 +/+ RUNX1-GFP or Fbxo11 +/– RUNX1-GFP transplants. n = 7–10 mice per group. **** P < 0.0001, by fixed-effects (type III) analysis. ( J ) Percentage of GFP + mononuclear cells isolated from BM aspirates of transplant recipients at 11 weeks. n = 8–9 mice per group. * P -linear < 0.05, by 2-tailed t test. ( K ) Percentage contribution to the LSK, Lin - /Sca1 + /Kit + /SLAM + (signaling lymphocyte activation molecules) (LSK-SLAM + ) gate of immunophenotypically defined HSPCs in surviving RUNX1 transplant recipients. n = 6 mice per group. LT-HSC, long-term HSC; ST-HSC, short-term HSC; MPP-GM, granulocyte-monocyte biased multipotent progenitors; MPP-MkE, megakaryocyte-erythroid biased multipotent progenitors; MPP Ly, lymphoid-biased multipotent progenitor. Q > 0.05, by t tests corrected for multiple comparisons (no significant differences were detected). ( L ) Percentage contribution of RUNX1-GFP + common myeloid progenitor (CMP), GMP, and megakaryocyte-erythrocyte progenitor (MEPs) in the Lin – c-kit + HSPC compartments. n = 6 mice per group. * P < 0.05, by multiple unpaired tests, t test for groups with normal distribution, or Mann-Whitney U test. ( M ) Differential CBCs in mice 16 weeks after pIpC. * P < 0.05, by unpaired, 2-tailed t for groups with normal distribution or Mann-Whitney U test. ( N ) Strategy of Nup98-Hoxd13 -driven MDS mouse model with sh CTRL or sh Fbxo11 vectors. n = 6–10 mice per group. ( O ) Western blot for FBXO11, NPM1, and ACTIN in c-kit + cells from Nup98-Hoxd13 + mice, transduced with lentiviral sh CTRL or sh Fbxo11 . ( P ) CBC in Nup98-Hoxd13 transplants at 2 months. sh Fbxo11 groups were pooled. * P < 0.05 and ** P < 0.005, by unpaired, 2-tailed t test for groups with normal distribution or Mann-Whitney U test. ( Q ) BM cellularity of Nup98-Hoxd13 mice. sh Fbxo11 groups were pooled. * P < 0.05 and ** P < 0.01, by Mann-Whitney U test. ( R ) Representative H&E-stained femur cells from Q . Original magnification, ×10. Scale bar: 200 μm.

Article Snippet: Human lentiviral shRNA constructs targeting FBXO11 in the pLVRU6MP-mCherry vectors were purchased from Genecopoeia.

Techniques: Western Blot, Double Knockout, Plasmid Preparation, Isolation, Activation Assay, MANN-WHITNEY, Transduction, Staining

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: ( A ) Map of FBXO11 mutations identified by whole-exome sequencing of the MSK-IMPACT cohort of patients with hematologic malignancies. ( B ) Alpha-fold predicted structure of FBXO11 with structural features and mutations within myeloid diseases mapped in the disordered N-terminus. ( C ) Variant allele frequencies plotted along the amino acid residues affected by FBXO11 mutations; FBXO11 functional domains are boxed. N-term., N-terminus; ZnF-UBR, zinc finger-ubiquitin-protein ligase E3 component n-Recognin 1. ( D ) Prediction of intrinsically unstructured proteins 2 (IUPRED2) score of amino acid residues in FBXO11-long. The blue arrowhead indicates the initial methionine residue in FBXO11-short. ( E ) Representative confocal images of FBXO11-long and FBXO11-short expressed in HEK293T cells. Scale bars: 10 μm. ( F ) Quantification of signal distribution of FBXO11-long + and FBXO11-short + cells using the SD of FLAG-FBXO11 signal across each nucleus. ** P < 0.01, by 2-tailed, unpaired Mann-Whitney U test. ( G ) GSEA analysis of RNA-Seq data ( GSE156708 ) from MDS-L cells that were FBXO11-KO compared with MDS-L parental cells, cells with reexpression of FBXO11-long cDNA, or cells with reexpression of FBXO11-short cDNA. The size of circle indicates the number of genes in the set.

Article Snippet: Human lentiviral shRNA constructs targeting FBXO11 in the pLVRU6MP-mCherry vectors were purchased from Genecopoeia.

Techniques: Sequencing, Variant Assay, Functional Assay, Ubiquitin Proteomics, Residue, MANN-WHITNEY, RNA Sequencing

Journal: The Journal of Clinical Investigation

Article Title: FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

doi: 10.1172/JCI193636

Figure Lengend Snippet: Patient characteristics associated with FBXO11 mutations identified in myeloid malignancies

Article Snippet: Human lentiviral shRNA constructs targeting FBXO11 in the pLVRU6MP-mCherry vectors were purchased from Genecopoeia.

Techniques: