Journal: Cancer cell

Article Title: Inherited and somatic defects in DDX41 in myeloid neoplasms

doi: 10.1016/j.ccell.2015.03.017

Figure Lengend Snippet: (A, B, C) Pedigrees of family 1 (A), family 2 (B), and family 3 (C) (upper panels). Age of diagnosis and detected DDX41 mutations are indicated. Lower panels show sequencing reads from whole exome sequencing (WES) with frequencies of detected mutations in bone marrow (BM) and germline samples. Confirmation of germline and somatic DDX41 mutations by Sanger sequencing is exemplarily shown for the family 2 (B). Arrows and bars indicated the specific nucleotide and predicted codon, respectively. Case number is annotated according to Table 1. Asymptomatic/presymptomatic carrier-clinically unaffected at this time but could later exhibit symptoms (Bennett et al., 2008)

Article Snippet: Primers and probes for all genes analyzed were purchased from Applied Biosystems gene expression assays products ( DDX41 : Hs00169602_m1; GAPDH : Hs99999905_m1).

Techniques: Biomarker Discovery, Sequencing

Journal: Cancer cell

Article Title: Inherited and somatic defects in DDX41 in myeloid neoplasms

doi: 10.1016/j.ccell.2015.03.017

Figure Lengend Snippet: Characteristics of DDX41 mutants.

Article Snippet: Primers and probes for all genes analyzed were purchased from Applied Biosystems gene expression assays products ( DDX41 : Hs00169602_m1; GAPDH : Hs99999905_m1).

Techniques: Mutagenesis

Journal: Cancer cell

Article Title: Inherited and somatic defects in DDX41 in myeloid neoplasms

doi: 10.1016/j.ccell.2015.03.017

Figure Lengend Snippet: (A) DDX41 is located at the distal end of chromosome 5q, 5q35.3, and encodes a protein that contains three known domains and ATP binding sites, as illustrated. The pink bars visualize deletions of chromosome 5q in our MDS cohort that include the DDX41 locus. The red triangles indicate DDX41 mutations in patients with hematological malignancies from our cohort and TCGA. Red circles indicate the identified germline mutations of DDX41 (p.Q52fs, p.D140fs, p.M155I and p.I396T). The p.R525H mutation was detected in 13 out of 1,045 cases. Purple triangles show DDX41 mutations in non-hematological malignancies. Sanger sequencing confirming recurrent germline mutation (p.D140fs; left) and somatic mutation (p.R525H; right) of DDX41 are shown.

Article Snippet: Primers and probes for all genes analyzed were purchased from Applied Biosystems gene expression assays products ( DDX41 : Hs00169602_m1; GAPDH : Hs99999905_m1).

Techniques: Binding Assay, Mutagenesis, Sequencing

Journal: Cancer cell

Article Title: Inherited and somatic defects in DDX41 in myeloid neoplasms

doi: 10.1016/j.ccell.2015.03.017

Figure Lengend Snippet: (A) Patients with somatic DDX41 defects (mutations and deletions) in different types of myeloid neoplasms. Indicated is the percentage of patients of each cohort with DDX41 deletions and mutations. The absolute number of patients with alterations is shown on the top of each bar.

Article Snippet: Primers and probes for all genes analyzed were purchased from Applied Biosystems gene expression assays products ( DDX41 : Hs00169602_m1; GAPDH : Hs99999905_m1).

Techniques:

Journal: Cancer cell

Article Title: Inherited and somatic defects in DDX41 in myeloid neoplasms

doi: 10.1016/j.ccell.2015.03.017

Figure Lengend Snippet: (A) Expression level of DDX41 in leukemic cell lines K562 and U937 and primary CD34+ cells as determined by normalization to GAPDH upon knockdown or overexpression of DDX41.

Article Snippet: Primers and probes for all genes analyzed were purchased from Applied Biosystems gene expression assays products ( DDX41 : Hs00169602_m1; GAPDH : Hs99999905_m1).

Techniques: Expressing, Knockdown, Over Expression

Journal: Cancer cell

Article Title: Inherited and somatic defects in DDX41 in myeloid neoplasms

doi: 10.1016/j.ccell.2015.03.017

Figure Lengend Snippet: (A) DDX41 interactions with spliceosomal protein complexes are indicated. Spliceosomal proteins that coimmunoprecipitated with DDX41 were organized in colored functional protein complexes based on Ingenuity pathway analysis and published data (Hegele et al., 2012). Individual protein enrichment was presented as total spectral counts and displayed by different circle size. Increased circle size indicates higher number of total spectra counts for the protein. Total spectral count is a semi-quantitative method to predict abundance of a specific protein and is not used to compare with abundance of other proteins. Unfilled double ring symbols indicate proteins that were not identified in DDX41 co-immunoprecipitation experiments, but which have been linked to the spliceosome.

Article Snippet: Primers and probes for all genes analyzed were purchased from Applied Biosystems gene expression assays products ( DDX41 : Hs00169602_m1; GAPDH : Hs99999905_m1).

Techniques: Functional Assay, Protein Enrichment, Immunoprecipitation

Journal: Cancer cell

Article Title: Inherited and somatic defects in DDX41 in myeloid neoplasms

doi: 10.1016/j.ccell.2015.03.017

Figure Lengend Snippet: (A) Increased exon skipping (top) and retention (bottom) in patients with DDX41 defects are indicated by an excess of green reads and red reads, respectively. The center panel shows a scatter plot of exon skipping in RNA isolated from control cells versus RNA from DDX41 defective mutant cells. Lines show the 10% difference cutoff limit used to select the most frequently affected exons.

Article Snippet: Primers and probes for all genes analyzed were purchased from Applied Biosystems gene expression assays products ( DDX41 : Hs00169602_m1; GAPDH : Hs99999905_m1).

Techniques: Isolation, Control, Mutagenesis

Journal: Leukemia

Article Title: Discriminating activities of DEAD-Box Helicase 41 from myeloid malignancy-associated germline variants by genetic rescue

doi: 10.1038/s41375-022-01753-4

Figure Lengend Snippet: A K331del and R293H pedigrees. Probands are indicated with an arrow. B Domain structure of human DDX41. Germline and somatic mutations are indicated within the corresponding domains. C Sanger sequencing data of probands in each family. D Predicted structure of mutant human DDX41 by AlphaFold7 and 3-D images are created using Mol* viewer (https://molstar.org/viewer/). The residues forming the hydrogen bond and surrounding residues are indicated in black. Residues where this interaction is lost due to mutation are indicated in red. E Confidence of predicted structure from AlphaFold analysis.

Article Snippet: Reagent Source Identifier Bacterial strains JM109 competent cells Promega L2001 Experimental model: Cell lines Mouse: hi- Ddx41 +/+ and hi- Ddx41 +/− cells This paper N/A CRISPR/Cas-9 system Predesigned Alt-R CRISPR-Cas9 gRNA for mouse Ddx41 Integrated DNA Technologies Mm.Cas9.DDX41.1.AA, Mm.Cas9.DDX41.1.AB, Mm.Cas9.DDX41.1.AC sNLS-spCas9-sNLS Aldevron 9212 Antibodies Flag-tag rabbit mAb Cell Signaling Technology 14793 Alexa Fluor 594 chicken anti-rabbit IgG (H+L) Invitrogen A21442 DDX41 mouse mAb Novus Biologicals H00051428-M01 DDX41 rabbit pAb Cocalico Biologicals, INC. Bresnick laboratory-generated β-actin mouse mAb Cell Signaling Technology 3700S GAPDH rabbit mAb Cell Signaling Technology 2118 Recombinant DNA pMSCV-PIG Mitchell Weiss (St. Jude Children’s Research Hospital) N/A pMSCV-PIG-DDX41 WT This paper N/A pMSCV-PIG-DDX41-G173R pMSCV-PIG-DDX41-R293H pMSCV-PIG-DDX41-K331del pMSCV-PIG-DDX41-R525H This paper N/A Chemicals Pierce TM ECL 2 Western Blotting Substrate Thermo Fisher Scientific 80196 SuperSignal TM West Femto Maximum Sensitivity Substrate Thermo Fisher Scientific 34096 Power SYBR Green Master Mix Applied Biosystems 4367659 TRIzol Reagent Thermo Fisher Scientific 15596 Phusion High-Fidelity DNA Polymerase Thermo Fisher Scientific F530S P3 Primary Cell 4D-Nucleofector ® X Kit Lonza V4XP-3032 Critical commercial kit RNAeasy Micro Kit Qiagen 74004 QIAquick Gel Extraction Kit Qiagen 28706 QIAquick PCR Purification Kit Qiagen 28106 Software Image Studio Lite (version 5.2) Image studio acquisition software https://www.licor.com/ Prism 9 GraphPad https://www.graphpad.com/ SnapGene viewer Dotmatics https://www.snapgene.com/snapgene-viewer/ Open in a separate window Key Resource Table.

Techniques: Sequencing, Mutagenesis

Journal: Leukemia

Article Title: Discriminating activities of DEAD-Box Helicase 41 from myeloid malignancy-associated germline variants by genetic rescue

doi: 10.1038/s41375-022-01753-4

Figure Lengend Snippet: A Genetic rescue system. Three CRISPR/Cas9 RNP complexes for Ddx41 were electroporated individually into HoxB-immortalized (hi)-progenitors, and clonal lines were isolated. Retrovirus expressing GFP or GFP and DDX41 or a human disease-associated DDX41 variant were infected into hi-Ddx41+/+ and hi-Ddx41+/− cells. GFP-positive cells were isolated by flow cytometry and analyzed by qRT-PCR and RNA-seq. Protein was analyzed by semi-quantitative western blotting. B Exons corresponding to representative domains and target loci of crRNAs on murine Ddx41. C Sanger sequencing of genomic DNA at the edited region of hi-Ddx41+/− clonal lines. The crRNA used to generate each clone is indicated in parenthesis. D DDX41 protein levels in hi-Ddx41+/+ and hi-Ddx41+/− cells were analyzed by semi-quantitative Western blotting using two different antibodies (mouse monoclonal anti-DDX41 antibody (2F4), Novus Biologicals, and our rabbit polyclonal anti-DDX41 antibody). Each clone number indicated corresponds to each clone in Fig. 2C. E Representative Western blots of Flag-tagged DDX41 and variants and endogenous DDX41 using anti-DDX41 antibody in genetic rescue assay. F Quantitative analysis of DDX41 protein of Fig. 2E (n=3 per group). G Subcellular localization of DDX41 and variants by confocal fluorescence microscope using anti-Flag antibody. H mRNAs regulated by DDX41 genetic rescue in hi-Ddx41+/− cells. Heatmap with TPM values of DDX41-regulated mRNAs from total RNA-Seq (n=3 per group). I qRT-PCR analysis of DDX41-regulated mRNAs (n=6 per group). Statistics were calculated using one-way ANOVA, followed by Tukey’s test; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. J Monocytic and granulocytic populations of live GFP+ cells with CD11b and CD115 surface markers detected by flow cytometry (n=6 per group). Statistics were calculated using one-way ANOVA, followed by Tukey’s test; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Article Snippet: Reagent Source Identifier Bacterial strains JM109 competent cells Promega L2001 Experimental model: Cell lines Mouse: hi- Ddx41 +/+ and hi- Ddx41 +/− cells This paper N/A CRISPR/Cas-9 system Predesigned Alt-R CRISPR-Cas9 gRNA for mouse Ddx41 Integrated DNA Technologies Mm.Cas9.DDX41.1.AA, Mm.Cas9.DDX41.1.AB, Mm.Cas9.DDX41.1.AC sNLS-spCas9-sNLS Aldevron 9212 Antibodies Flag-tag rabbit mAb Cell Signaling Technology 14793 Alexa Fluor 594 chicken anti-rabbit IgG (H+L) Invitrogen A21442 DDX41 mouse mAb Novus Biologicals H00051428-M01 DDX41 rabbit pAb Cocalico Biologicals, INC. Bresnick laboratory-generated β-actin mouse mAb Cell Signaling Technology 3700S GAPDH rabbit mAb Cell Signaling Technology 2118 Recombinant DNA pMSCV-PIG Mitchell Weiss (St. Jude Children’s Research Hospital) N/A pMSCV-PIG-DDX41 WT This paper N/A pMSCV-PIG-DDX41-G173R pMSCV-PIG-DDX41-R293H pMSCV-PIG-DDX41-K331del pMSCV-PIG-DDX41-R525H This paper N/A Chemicals Pierce TM ECL 2 Western Blotting Substrate Thermo Fisher Scientific 80196 SuperSignal TM West Femto Maximum Sensitivity Substrate Thermo Fisher Scientific 34096 Power SYBR Green Master Mix Applied Biosystems 4367659 TRIzol Reagent Thermo Fisher Scientific 15596 Phusion High-Fidelity DNA Polymerase Thermo Fisher Scientific F530S P3 Primary Cell 4D-Nucleofector ® X Kit Lonza V4XP-3032 Critical commercial kit RNAeasy Micro Kit Qiagen 74004 QIAquick Gel Extraction Kit Qiagen 28706 QIAquick PCR Purification Kit Qiagen 28106 Software Image Studio Lite (version 5.2) Image studio acquisition software https://www.licor.com/ Prism 9 GraphPad https://www.graphpad.com/ SnapGene viewer Dotmatics https://www.snapgene.com/snapgene-viewer/ Open in a separate window Key Resource Table.

Techniques: CRISPR, Isolation, Expressing, Variant Assay, Infection, Flow Cytometry, Quantitative RT-PCR, RNA Sequencing, Western Blot, Sequencing, Rescue Assay, Fluorescence, Microscopy

Journal: Leukemia

Article Title: Discriminating activities of DEAD-Box Helicase 41 from myeloid malignancy-associated germline variants by genetic rescue

doi: 10.1038/s41375-022-01753-4

Figure Lengend Snippet: Primer sequences

Article Snippet: Reagent Source Identifier Bacterial strains JM109 competent cells Promega L2001 Experimental model: Cell lines Mouse: hi- Ddx41 +/+ and hi- Ddx41 +/− cells This paper N/A CRISPR/Cas-9 system Predesigned Alt-R CRISPR-Cas9 gRNA for mouse Ddx41 Integrated DNA Technologies Mm.Cas9.DDX41.1.AA, Mm.Cas9.DDX41.1.AB, Mm.Cas9.DDX41.1.AC sNLS-spCas9-sNLS Aldevron 9212 Antibodies Flag-tag rabbit mAb Cell Signaling Technology 14793 Alexa Fluor 594 chicken anti-rabbit IgG (H+L) Invitrogen A21442 DDX41 mouse mAb Novus Biologicals H00051428-M01 DDX41 rabbit pAb Cocalico Biologicals, INC. Bresnick laboratory-generated β-actin mouse mAb Cell Signaling Technology 3700S GAPDH rabbit mAb Cell Signaling Technology 2118 Recombinant DNA pMSCV-PIG Mitchell Weiss (St. Jude Children’s Research Hospital) N/A pMSCV-PIG-DDX41 WT This paper N/A pMSCV-PIG-DDX41-G173R pMSCV-PIG-DDX41-R293H pMSCV-PIG-DDX41-K331del pMSCV-PIG-DDX41-R525H This paper N/A Chemicals Pierce TM ECL 2 Western Blotting Substrate Thermo Fisher Scientific 80196 SuperSignal TM West Femto Maximum Sensitivity Substrate Thermo Fisher Scientific 34096 Power SYBR Green Master Mix Applied Biosystems 4367659 TRIzol Reagent Thermo Fisher Scientific 15596 Phusion High-Fidelity DNA Polymerase Thermo Fisher Scientific F530S P3 Primary Cell 4D-Nucleofector ® X Kit Lonza V4XP-3032 Critical commercial kit RNAeasy Micro Kit Qiagen 74004 QIAquick Gel Extraction Kit Qiagen 28706 QIAquick PCR Purification Kit Qiagen 28106 Software Image Studio Lite (version 5.2) Image studio acquisition software https://www.licor.com/ Prism 9 GraphPad https://www.graphpad.com/ SnapGene viewer Dotmatics https://www.snapgene.com/snapgene-viewer/ Open in a separate window Key Resource Table.

Techniques: Sequencing

Journal: Leukemia

Article Title: Discriminating activities of DEAD-Box Helicase 41 from myeloid malignancy-associated germline variants by genetic rescue

doi: 10.1038/s41375-022-01753-4

Figure Lengend Snippet: Key Resource Table

Article Snippet: Reagent Source Identifier Bacterial strains JM109 competent cells Promega L2001 Experimental model: Cell lines Mouse: hi- Ddx41 +/+ and hi- Ddx41 +/− cells This paper N/A CRISPR/Cas-9 system Predesigned Alt-R CRISPR-Cas9 gRNA for mouse Ddx41 Integrated DNA Technologies Mm.Cas9.DDX41.1.AA, Mm.Cas9.DDX41.1.AB, Mm.Cas9.DDX41.1.AC sNLS-spCas9-sNLS Aldevron 9212 Antibodies Flag-tag rabbit mAb Cell Signaling Technology 14793 Alexa Fluor 594 chicken anti-rabbit IgG (H+L) Invitrogen A21442 DDX41 mouse mAb Novus Biologicals H00051428-M01 DDX41 rabbit pAb Cocalico Biologicals, INC. Bresnick laboratory-generated β-actin mouse mAb Cell Signaling Technology 3700S GAPDH rabbit mAb Cell Signaling Technology 2118 Recombinant DNA pMSCV-PIG Mitchell Weiss (St. Jude Children’s Research Hospital) N/A pMSCV-PIG-DDX41 WT This paper N/A pMSCV-PIG-DDX41-G173R pMSCV-PIG-DDX41-R293H pMSCV-PIG-DDX41-K331del pMSCV-PIG-DDX41-R525H This paper N/A Chemicals Pierce TM ECL 2 Western Blotting Substrate Thermo Fisher Scientific 80196 SuperSignal TM West Femto Maximum Sensitivity Substrate Thermo Fisher Scientific 34096 Power SYBR Green Master Mix Applied Biosystems 4367659 TRIzol Reagent Thermo Fisher Scientific 15596 Phusion High-Fidelity DNA Polymerase Thermo Fisher Scientific F530S P3 Primary Cell 4D-Nucleofector ® X Kit Lonza V4XP-3032 Critical commercial kit RNAeasy Micro Kit Qiagen 74004 QIAquick Gel Extraction Kit Qiagen 28706 QIAquick PCR Purification Kit Qiagen 28106 Software Image Studio Lite (version 5.2) Image studio acquisition software https://www.licor.com/ Prism 9 GraphPad https://www.graphpad.com/ SnapGene viewer Dotmatics https://www.snapgene.com/snapgene-viewer/ Open in a separate window Key Resource Table.

Techniques: CRISPR, Recombinant, Western Blot, SYBR Green Assay, Gel Extraction, Purification, Software

Journal: Leukemia

Article Title: Discriminating activities of DEAD-Box Helicase 41 from myeloid malignancy-associated germline variants by genetic rescue

doi: 10.1038/s41375-022-01753-4

Figure Lengend Snippet: A K331del and R293H pedigrees. Probands are indicated with an arrow. B Domain structure of human DDX41. Germline and somatic mutations are indicated within the corresponding domains. C Sanger sequencing data of probands in each family. D Predicted structure of mutant human DDX41 by AlphaFold7 and 3-D images are created using Mol* viewer (https://molstar.org/viewer/). The residues forming the hydrogen bond and surrounding residues are indicated in black. Residues where this interaction is lost due to mutation are indicated in red. E Confidence of predicted structure from AlphaFold analysis.

Article Snippet: D DDX41 protein levels in hi- Ddx41 +/+ and hi- Ddx41 +/− cells were analyzed by semi-quantitative Western blotting using two different antibodies (mouse monoclonal anti-DDX41 antibody (2F4), Novus Biologicals, and our rabbit polyclonal anti-DDX41 antibody).

Techniques: Sequencing, Mutagenesis

Journal: Leukemia

Article Title: Discriminating activities of DEAD-Box Helicase 41 from myeloid malignancy-associated germline variants by genetic rescue

doi: 10.1038/s41375-022-01753-4

Figure Lengend Snippet: A Genetic rescue system. Three CRISPR/Cas9 RNP complexes for Ddx41 were electroporated individually into HoxB-immortalized (hi)-progenitors, and clonal lines were isolated. Retrovirus expressing GFP or GFP and DDX41 or a human disease-associated DDX41 variant were infected into hi-Ddx41+/+ and hi-Ddx41+/− cells. GFP-positive cells were isolated by flow cytometry and analyzed by qRT-PCR and RNA-seq. Protein was analyzed by semi-quantitative western blotting. B Exons corresponding to representative domains and target loci of crRNAs on murine Ddx41. C Sanger sequencing of genomic DNA at the edited region of hi-Ddx41+/− clonal lines. The crRNA used to generate each clone is indicated in parenthesis. D DDX41 protein levels in hi-Ddx41+/+ and hi-Ddx41+/− cells were analyzed by semi-quantitative Western blotting using two different antibodies (mouse monoclonal anti-DDX41 antibody (2F4), Novus Biologicals, and our rabbit polyclonal anti-DDX41 antibody). Each clone number indicated corresponds to each clone in Fig. 2C. E Representative Western blots of Flag-tagged DDX41 and variants and endogenous DDX41 using anti-DDX41 antibody in genetic rescue assay. F Quantitative analysis of DDX41 protein of Fig. 2E (n=3 per group). G Subcellular localization of DDX41 and variants by confocal fluorescence microscope using anti-Flag antibody. H mRNAs regulated by DDX41 genetic rescue in hi-Ddx41+/− cells. Heatmap with TPM values of DDX41-regulated mRNAs from total RNA-Seq (n=3 per group). I qRT-PCR analysis of DDX41-regulated mRNAs (n=6 per group). Statistics were calculated using one-way ANOVA, followed by Tukey’s test; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. J Monocytic and granulocytic populations of live GFP+ cells with CD11b and CD115 surface markers detected by flow cytometry (n=6 per group). Statistics were calculated using one-way ANOVA, followed by Tukey’s test; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Article Snippet: D DDX41 protein levels in hi- Ddx41 +/+ and hi- Ddx41 +/− cells were analyzed by semi-quantitative Western blotting using two different antibodies (mouse monoclonal anti-DDX41 antibody (2F4), Novus Biologicals, and our rabbit polyclonal anti-DDX41 antibody).

Techniques: CRISPR, Isolation, Expressing, Variant Assay, Infection, Flow Cytometry, Quantitative RT-PCR, RNA Sequencing Assay, Western Blot, Sequencing, Rescue Assay, Fluorescence, Microscopy

Journal: Leukemia

Article Title: Discriminating activities of DEAD-Box Helicase 41 from myeloid malignancy-associated germline variants by genetic rescue

doi: 10.1038/s41375-022-01753-4

Figure Lengend Snippet: Primer sequences

Article Snippet: D DDX41 protein levels in hi- Ddx41 +/+ and hi- Ddx41 +/− cells were analyzed by semi-quantitative Western blotting using two different antibodies (mouse monoclonal anti-DDX41 antibody (2F4), Novus Biologicals, and our rabbit polyclonal anti-DDX41 antibody).

Techniques: Sequencing

Journal: Leukemia

Article Title: Discriminating activities of DEAD-Box Helicase 41 from myeloid malignancy-associated germline variants by genetic rescue

doi: 10.1038/s41375-022-01753-4

Figure Lengend Snippet: Key Resource Table

Article Snippet: D DDX41 protein levels in hi- Ddx41 +/+ and hi- Ddx41 +/− cells were analyzed by semi-quantitative Western blotting using two different antibodies (mouse monoclonal anti-DDX41 antibody (2F4), Novus Biologicals, and our rabbit polyclonal anti-DDX41 antibody).

Techniques: CRISPR, Recombinant, Western Blot, SYBR Green Assay, Gel Extraction, Purification, Software