Journal: Oncology Letters

Article Title: DEK is highly expressed in breast cancer and is associated with malignant phenotype and progression

doi: 10.3892/ol.2021.12701

Figure Lengend Snippet: Expression of β-catenin and Wnt target proteins following overexpression and knockdown of DEK in breast cancer cells. (A and C) Representative western blotting bands and (B and D) quantification of protein expression in MCF7 cells. GAPDH served as an internal control. *P<0.05 DEK vs. NC or shDEK vs. shNC, respectively. DEK, DEK proto-oncogene; Gsk-3β, glycogen synthase kinase-3β; NC, negative control; sh, short hairpin.

Article Snippet: The membranes were incubated with primary antibodies against DEK (cat. no. 16448-1-AP; 1:1,000; ProteinTech Group, Inc.), glycogen synthase kinase-3β (Gsk-3β; cat. no. 5676; 1:1,000; Cell Signaling Technology, Inc.), cyclin D1 (cat. no. SC-8396; 1:100; Santa Cruz Biotechnology, Inc.), β-catenin (cat. no. 17565-1-AP), active β-catenin (cat. no. 51067-2-AP), c-Myc (cat. no. 67447-1-Ig) and GAPDH (cat. no. 60004-1-Ig) (all 1:1,000; ProteinTech Group, Inc.) overnight at 4°C.

Techniques: Expressing, Over Expression, Western Blot, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: Nuclear DEK preserves hematopoietic stem cells potential via NCoR1/HDAC3-Akt1/2-mTOR axis

doi: 10.1084/jem.20201974

Figure Lengend Snippet: Akt1 and Akt2 are direct targets of DEK in HSCs by altering the chromatin accessibility landscape. (A) Representative heatmap of genome-wide ATAC-seq signal around genes in HSCs freshly sorted from Dek fl/fl and Dek-cKO mice ( n = 3 biological independent samples per group). (B) Average diagram of genome-wide chromatin accessibility at TSS regions (±2,000 bp). (C) Location of all ATAC-seq peaks in Dek fl/fl and Dek-cKO HSCs. Promoter-TSS, the region between promoter and TSS (−1 kb to 100 bp). (D) Enrichment of the increased known TF binding motifs in different chromatin-accessible regions. (E) Enrichment of the decreased known TF binding motifs in different chromatin-accessible regions. (F) Pathway analysis of the genes with significant changed ATAC peaks (over twofold change; P < 0.01) in DEK -deficient HSCs. (G) Integrative analysis to identify transcriptome-wide potential targets of DEK in HSCs. Left: Potential negative targets of DEK. Right: Potential positive targets of DEK. RNA-Up and RNA-Down indicate genes with significantly increased and decreased expression, respectively, upon DEK deletion in HSCs as detected by RNA-seq (FPKM > 1, fold change > 1.5). CUT&Tag indicates genes with significant enrichment in DEK binding (reads per kilobase per million > 1). Accessibility-Increased and Accessibility-Decreased indicates genes with significantly increased and decreased accessibility, respectively, upon DEK deletion in HSCs as detected by ATAC-seq (reads per kilobase per million > 1, fold change > 2). (H) Summary of the PI3K-Akt-mTOR pathway. (I) Accessible chromatin located at gene loci, including Pi3kr1 , Akt1 , Akt2 , and mTOR . (J) The FPKM value of indicated genes. Data are from RNA-seq. (K) Relative mRNA expression of Akt1 and Akt2 in freshly sorted Dek fl/fl and Dek-cKO HSCs ( n = 4). (L) FACS analysis of p-Akt in HSCs of the indicated mice. The histograms indicate the mean fluorescence intensity analysis ( n = 4). (M) Western blot of PI3K-Akt-mTOR pathway proteins in lysates prepared from freshly sorted Lin − c-Kit + cells. **, P < 0.01; ***, P < 0.001; Student’s t test. Data in K–M are representative of three independent experiments. VEGF, vascular endothelial growth factor.

Article Snippet: The antibodies used in this study were against the following: DEK (1:1,000; 16448–1-AP; Proteintech), DEK (1:1,000; 66194–1-Ig; Proteintech), PI3K (1:1,000; AF1549; Beyotime), p-PI3K (1:1,000; 4228T; Cell Signaling Technology), Akt (1:1,000; 10176–2-AP; Proteintech), p-Akt (1:1,000; 66444–1-Ig; Proteintech), mTOR (1:1,000; 20657–1-AP; Proteintech), p-mTOR (1:1,000; 610301; BioLegend), Snf2h (1:1,000; 13066–1-AP; Proteintech), NCoR1 (1:1,000; sc-515934; Santa Cruz Biotechnology), HDAC3 (1:1,000; sc-376957; Santa Cruz Biotechnology), HDAC3 (1:1,000; 10255–1-AP; Proteintech), mono-methyl-histone H3 (Lys4; 1:1,000; 5326T; Cell Signaling Technology), di-methyl-histone H3 (Lys4; 1:1,000; 9725T; Cell Signaling Technology), H3K4me3 (1:1,000; 9751T; Cell Signaling Technology), di-methyl-histone H3 (Lys9; 1:1,000; AH438; Beyotime), tri-methyl-histone H3 (Lys9; 1:1,000; 13969T; Cell Signaling Technology), tri-methyl-histone H3 (Lys27; 1:1,000; 9733S; Cell Signaling Technology), H3K9ac (1:1,000; 9649T; Cell Signaling Technology), acetyl-histone H3 (Lys18; 1:1,000; 9675T; Cell Signaling Technology), H3K27ac (1:1,000; 8173T; Cell Signaling Technology), tubulin (1:1,000; AF0001; Beyotime), Lamin B1 (1:1,000; AF1408; Beyotime), H3 (1:1,000; AH433; Beyotime), H2A (1:1,000; AH419; Beyotime), H2B (1:1,000; AH426; Beyotime), and H4 (1:1,000; AH458; Beyotime).

Techniques: Genome Wide, Binding Assay, Expressing, RNA Sequencing Assay, Fluorescence, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Nuclear DEK preserves hematopoietic stem cells potential via NCoR1/HDAC3-Akt1/2-mTOR axis

doi: 10.1084/jem.20201974

Figure Lengend Snippet: DEK induces deacetylation of H3K27 by recruiting the corepressor NCoR1. (A) Western blot for modified histone 3 in lysates prepared from freshly sorted Lin − c-Kit + cells. (B and C) FACS analysis of H3K27ac level in HSCs (CD34 − LSK). The histograms indicate the mean fluorescence intensity (MFI) analysis of H3K27ac in HSCs ( n = 3). (D) Representative heatmap of genome-wide H3K27ac CUT&Tag signal around genes. (E) Average diagram of genome-wide H3K27ac CUT&Tag peaks at TSS regions (±3,000 bp). (F) Correlation of changes between ATAC peaks and H3K27ac CUT&Tag peaks. Correlation coefficient ( r ) and P values ( r = 0.72, P < 1 × 10 −50 ) were calculated by Pearson’s correlation analysis. (G) Distribution of ATAC peaks and H3K27ac CUT&Tag peaks across the indicated gene loci. (H) Schematic representation of the workflow for DEK partners’ discovery. Potential DEK interacting factors from published mass spectrometry data . The highly expressed genes in HSCs have an FPKM value of >30 (data from RNA-seq). (I) In situ ligation assay to detect DEK/Snf2h and DEK/NCoR1 interaction. As a negative control, proximity ligation was performed using a rabbit anti-DEK antibody and a mouse IgG. Nuclei were visualized using DAPI staining. Scale bar: 5 µm. (J) Lin − c-Kit + cells were freshly isolated from mice. DEK or NCoR1 protein was immunoprecipitated from cell lysates, followed by immunoblotting (IB). (K) Western blot for H3K27ac, DEK, and tubulin in lysates prepared from Lin − c-Kit + cells. Lin − c-Kit + cells were sorted and cultured in vitro, with treatment of RGFP-966 (5 µM) or GNE-049 (500 nM) for 24 h. (L) qRT-PCR analysis of the indicated transcripts from HSCs ( n = 4). HSCs were sorted and cultured in vitro, with treatment with RGFP-966 (5 µM) or GNE-049 (500 nM) for 24 h. Error bars represent means ± SD. *, P < 0.05, **, P < 0.01; Student’s t test or one-way ANOVA. Data in A–C and I–L are representative of three independent experiments. TES, transcriptional end site.

Article Snippet: The antibodies used in this study were against the following: DEK (1:1,000; 16448–1-AP; Proteintech), DEK (1:1,000; 66194–1-Ig; Proteintech), PI3K (1:1,000; AF1549; Beyotime), p-PI3K (1:1,000; 4228T; Cell Signaling Technology), Akt (1:1,000; 10176–2-AP; Proteintech), p-Akt (1:1,000; 66444–1-Ig; Proteintech), mTOR (1:1,000; 20657–1-AP; Proteintech), p-mTOR (1:1,000; 610301; BioLegend), Snf2h (1:1,000; 13066–1-AP; Proteintech), NCoR1 (1:1,000; sc-515934; Santa Cruz Biotechnology), HDAC3 (1:1,000; sc-376957; Santa Cruz Biotechnology), HDAC3 (1:1,000; 10255–1-AP; Proteintech), mono-methyl-histone H3 (Lys4; 1:1,000; 5326T; Cell Signaling Technology), di-methyl-histone H3 (Lys4; 1:1,000; 9725T; Cell Signaling Technology), H3K4me3 (1:1,000; 9751T; Cell Signaling Technology), di-methyl-histone H3 (Lys9; 1:1,000; AH438; Beyotime), tri-methyl-histone H3 (Lys9; 1:1,000; 13969T; Cell Signaling Technology), tri-methyl-histone H3 (Lys27; 1:1,000; 9733S; Cell Signaling Technology), H3K9ac (1:1,000; 9649T; Cell Signaling Technology), acetyl-histone H3 (Lys18; 1:1,000; 9675T; Cell Signaling Technology), H3K27ac (1:1,000; 8173T; Cell Signaling Technology), tubulin (1:1,000; AF0001; Beyotime), Lamin B1 (1:1,000; AF1408; Beyotime), H3 (1:1,000; AH433; Beyotime), H2A (1:1,000; AH419; Beyotime), H2B (1:1,000; AH426; Beyotime), and H4 (1:1,000; AH458; Beyotime).

Techniques: Western Blot, Modification, Fluorescence, Genome Wide, Mass Spectrometry, RNA Sequencing Assay, In Situ, Ligation, Negative Control, Staining, Isolation, Immunoprecipitation, Cell Culture, In Vitro, Quantitative RT-PCR

Journal: The Journal of Experimental Medicine

Article Title: Nuclear DEK preserves hematopoietic stem cells potential via NCoR1/HDAC3-Akt1/2-mTOR axis

doi: 10.1084/jem.20201974

Figure Lengend Snippet: Targeting the Akt-mTOR pathway partially rescues Dek-cKO defective HSCs. (A) Experimental schematic for administration of PI3K-mTOR pathway inhibitors. 1 mo after pIpC injection, Dek fl/fl and Dek fl/fl Mx1-Cre mice were orally gavaged with NVP-BEZ235 or MK-2206 for 2 mo. (B) FACS and mean fluorescence intensity (MFI) analysis of p-S6 in HSCs of Dek fl/fl Mx1-Cre mice at 2 mo after inhibitor treatment ( n = 3). (C) Glucose consumption of LSK cells sorted from the indicated mice. Cells were cultured in vitro for 24 h and the change in glucose concentration in the culture medium was measured ( n = 4). (D) ATP level in freshly sorted HSCs ( n = 4). (E) FACS analysis of Ki-67 + DAPI staining in LSK cells. (F and G) G0 phase analysis of LSK cells and HSCs in mice ( n = 4). (H–K) BM cell, HPC, LSK cell, and HSC count in mice BM ( n = 4–8). (L) Experimental schematic for the competitive transplantation assay. BM cells of Dek fl/fl and Dek - cKO mice were transplanted into lethally irradiated recipient mice with competitor BM cells from WT Ly5.1 mice. Recipient mice were subjected to inhibitor treatment at 1 mo after transplantation. The reconstituted BM cells of recipient mice were harvested (at 6 mo after transplantation) for analysis. (M) Percentage of donor-derived PB cells at the indicated time points in competitive transplantation assay ( n = 4–5). (N) Percentage of donor-derived BM cells, HPCs, LSK cells, and HSCs at 6 mo after first transplantation ( n = 4–5). (O) Experimental schematic for competitive transplantation assay. Lin − cells were freshly isolated from mice and subjected to retroviral transduction to enforce the expression of PTEN with GFP fluorescence. (P) FACS analysis of GFP + cells in donor-derived PB cells of recipient mice. (Q) Contribution of retrovirally transduced donor cells (CD45.2 + GFP + ) to recipient mouse PB cells after primary transplantation ( n = 5). (R) Proposed model demonstrating the role and underlying mechanisms of DEK regulating quiescence and metabolic hemostasis, and self-renewal of HSCs. Error bars represent means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; one-way ANOVA. Data are representative of three independent experiments.

Article Snippet: The antibodies used in this study were against the following: DEK (1:1,000; 16448–1-AP; Proteintech), DEK (1:1,000; 66194–1-Ig; Proteintech), PI3K (1:1,000; AF1549; Beyotime), p-PI3K (1:1,000; 4228T; Cell Signaling Technology), Akt (1:1,000; 10176–2-AP; Proteintech), p-Akt (1:1,000; 66444–1-Ig; Proteintech), mTOR (1:1,000; 20657–1-AP; Proteintech), p-mTOR (1:1,000; 610301; BioLegend), Snf2h (1:1,000; 13066–1-AP; Proteintech), NCoR1 (1:1,000; sc-515934; Santa Cruz Biotechnology), HDAC3 (1:1,000; sc-376957; Santa Cruz Biotechnology), HDAC3 (1:1,000; 10255–1-AP; Proteintech), mono-methyl-histone H3 (Lys4; 1:1,000; 5326T; Cell Signaling Technology), di-methyl-histone H3 (Lys4; 1:1,000; 9725T; Cell Signaling Technology), H3K4me3 (1:1,000; 9751T; Cell Signaling Technology), di-methyl-histone H3 (Lys9; 1:1,000; AH438; Beyotime), tri-methyl-histone H3 (Lys9; 1:1,000; 13969T; Cell Signaling Technology), tri-methyl-histone H3 (Lys27; 1:1,000; 9733S; Cell Signaling Technology), H3K9ac (1:1,000; 9649T; Cell Signaling Technology), acetyl-histone H3 (Lys18; 1:1,000; 9675T; Cell Signaling Technology), H3K27ac (1:1,000; 8173T; Cell Signaling Technology), tubulin (1:1,000; AF0001; Beyotime), Lamin B1 (1:1,000; AF1408; Beyotime), H3 (1:1,000; AH433; Beyotime), H2A (1:1,000; AH419; Beyotime), H2B (1:1,000; AH426; Beyotime), and H4 (1:1,000; AH458; Beyotime).

Techniques: Injection, Fluorescence, Cell Culture, In Vitro, Concentration Assay, Staining, Transplantation Assay, Irradiation, Derivative Assay, Isolation, Transduction, Expressing

Journal: STAR Protocols

Article Title: Protocol for Isolation and Characterization of In Situ Fixed Quiescent Muscle Stem Cells

doi: 10.1016/j.xpro.2020.100128

Figure Lengend Snippet:

Article Snippet: Anti-Dek (Optional) , ProteinTech Group , Catalog #: 16448-1-AP; RRID: AB_2092097.

Techniques: Recombinant, Electron Microscopy, Electrophoresis, Cell Culture, Software, RNA HS Assay