Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Transcription Factor Bach1 Suppresses the Developmental Angiogenesis of Zebrafish

doi: 10.1155/2017/2143875

Figure Lengend Snippet: The expression of Bach1 during embryonic development in zebrafish. Bach1 expression was evaluated in zebrafish embryos that had been stained with (a–c) or without (d–f) a Bach1 antibody at 4 h (a, d), 8 h (b, e), and 24 h (c, f) after fertilization. Scale bar: 100 μ m.

Article Snippet: SDS-PAGE was performed and proteins were detected using antibodies to Bach1 (sc-14700, Santa Cruz, CA) and β -actin (A5316, Sigma-Aldrich, St. Louis, MO).

Techniques: Expressing, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Transcription Factor Bach1 Suppresses the Developmental Angiogenesis of Zebrafish

doi: 10.1155/2017/2143875

Figure Lengend Snippet: Bach1 overexpression induces vascular defects in zebrafish embryos. (a) Zebrafish vasculature was evaluated via fluorescent images (C-D) or confocal images (E and F) in normal 32-hpf Tg(fli1:EGFP) zebrafish embryos (A, C and E) and in embryos that had been injected with 200 pg of Bach1 mRNA (B, D and F) at the single-cell stage. Bach1 mRNA injections disrupted formation of the intersegmental vessels (ISV) (arrow) and the dorsal longitudinal anastomotic vessels (DLAV) (asterisks). (A–D) Scale bar: 100 μ m. (E and F) Scale bar: 50 μ m. (b) Bach1 mRNA levels were evaluated in normal and Bach1-mRNA–injected embryos via quantitative real-time RT-PCR ( n = 8; ∗∗ P < 0.01 versus Control). (c) Embryos with defects in ISV and DLAV were counted and expressed as a percentage of the total number of embryos in each experimental group ( n = 50; ∗ P < 0.05 versus Control).

Article Snippet: SDS-PAGE was performed and proteins were detected using antibodies to Bach1 (sc-14700, Santa Cruz, CA) and β -actin (A5316, Sigma-Aldrich, St. Louis, MO).

Techniques: Over Expression, Injection, Quantitative RT-PCR, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Transcription Factor Bach1 Suppresses the Developmental Angiogenesis of Zebrafish

doi: 10.1155/2017/2143875

Figure Lengend Snippet: Bach1 suppresses endogenous Wnt/ β -catenin signaling and exogenous Wnt8a stimulated VEGF and IL-8 gene expression in zebrafish. (a) Representative fluorescent images of 36-hpf Tg(TOP:GFP) transgenic zebrafish embryos that had been injected with or without Bach1 mRNA. (b) The fluorescence intensity of GFP was quantified and expressed as the as the ratio of measurements Bach1 mRNA and Control group ( n = 12; ∗∗ P < 0.01 versus Control). (c) IL-8, VEGF 165 , and Bach1 mRNA levels were evaluated in 32-hpf Tg(hsp:wnt8a) embryos following Bach1 mRNA injections at the one cell stage and Wnt pathway activation (heat-shock) at 24 hpf ( n = 10; ∗ P < 0.05, ∗∗ P < 0.01 versus Control; ## P < 0.01 versus Wnt8a).

Article Snippet: SDS-PAGE was performed and proteins were detected using antibodies to Bach1 (sc-14700, Santa Cruz, CA) and β -actin (A5316, Sigma-Aldrich, St. Louis, MO).

Techniques: Gene Expression, Transgenic Assay, Injection, Fluorescence, Control, Activation Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Transcription Factor Bach1 Suppresses the Developmental Angiogenesis of Zebrafish

doi: 10.1155/2017/2143875

Figure Lengend Snippet: Bach1 occupies the TCF/LEF-binding site of the VEGF promoter and recruits HDAC1 to the VEGF promoter in HUVECs. (a) Potential TCF/LEF-binding site of the VEGF promoter is shown. Luciferase reporter constructs were created containing the truncated (−2650, −529, and −228) versions of the VEGF promoter; then the VEGF reporter or a pGL3-basic luciferase reporter and β -gal were cotransfected with a Bach1-coding vector or with an empty vector (Control) into HEK293T cells, and luciferase activity was evaluated 48 hours later ( n = 3; ∗∗ P < 0.01 versus Control). (b) Chromatin immunoprecipitation DNA was isolated with an anti-Bach1 antibody or with a Control IgG antibody, and then quantitative real-time RT-PCR analyses were performed with primer sequences from the indicated regions of the VEGF promoter in HUVECs ( n = 4; ∗ P < 0.05 versus Control IgG). (c) HUVECs were transfected with AdGFP or AdBach1; then chromatin immunoprecipitation was performed with antibodies against the indicated proteins. Quantitative real-time RT-PCR analyses were performed with a primer for the VEGF promoter (−301/−105) ( n = 3; ∗∗ P < 0.01 versus AdGFP). (d) HUVECs were transfected with AdGFP or AdBach1 and with the VEGF (−228) promoter construct; then, the cells were treated with or without TSA (0.1 or 0.5 μ mol/L) for 24 hours and luciferase activity was quantified ( n = 3; ∗ P < 0.05, ∗∗ P < 0.01).

Article Snippet: SDS-PAGE was performed and proteins were detected using antibodies to Bach1 (sc-14700, Santa Cruz, CA) and β -actin (A5316, Sigma-Aldrich, St. Louis, MO).

Techniques: Binding Assay, Luciferase, Construct, Plasmid Preparation, Control, Activity Assay, Chromatin Immunoprecipitation, Isolation, Quantitative RT-PCR, Transfection

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Transcription Factor Bach1 Suppresses the Developmental Angiogenesis of Zebrafish

doi: 10.1155/2017/2143875

Figure Lengend Snippet: Bach1 inhibits VEGF mRNA and protein expression in HUVECs. (a and b) mRNA or protein levels of VEGF 165 were compared in ConsiRNA- and Bach1siRNA-transfected HUVECs and in AdGFP and AdBach1-HUVECs. mRNA levels (a) were evaluated via quantitative PCR ( n = 4; ∗ P < 0.05) and protein levels (b) were evaluated via ELISA ( n = 3; ∗ P < 0.05).

Article Snippet: SDS-PAGE was performed and proteins were detected using antibodies to Bach1 (sc-14700, Santa Cruz, CA) and β -actin (A5316, Sigma-Aldrich, St. Louis, MO).

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Transcription Factor Bach1 Suppresses the Developmental Angiogenesis of Zebrafish

doi: 10.1155/2017/2143875

Figure Lengend Snippet: Exogenous administration of VEGF or IL-8 partially rescued Bach1-driven antiangiogenic response in HUVECs. (a) HUVECs were transfected with AdGFP or AdBach1 for 24 hours and then incubated with or without VEGF (50 ng/mL) or IL-8 (50 ng/mL) for 24 hours. Endothelial tube formation assay in Matrigel was performed in presence of VEGF or IL-8. Tube length was quantified and expressed as the fold change relative to AdGFP ( n = 4; ∗ P < 0.05, ∗∗ P < 0.01). Scale bar: 500 μ m. (b) Bach1 and β -actin protein levels were evaluated via Western blot.

Article Snippet: SDS-PAGE was performed and proteins were detected using antibodies to Bach1 (sc-14700, Santa Cruz, CA) and β -actin (A5316, Sigma-Aldrich, St. Louis, MO).

Techniques: Transfection, Incubation, Endothelial Tube Formation Assay, Western Blot

Journal: Cancer Management and Research

Article Title: CD24 expression and stem-associated features define tumor cell heterogeneity and tumorigenic capacities in a model of carcinogenesis

doi: 10.2147/CMAR.S176654

Figure Lengend Snippet: Xenograft-derived human embryonic kidney cells exhibit a heterogeneous gene expression of CD24. Notes: ( A ) CD44 + CD24 Pos and CD44 + CD24 Neg cells were sorted by FACS according to CD24 expression. ( B ) The purity of these two cell populations was confirmed by rerunning the sorted population (post-sorting). ( C ) Sorted cells were kept in culture for 1 month, after which cell surface marker expression was determined in both CD44 + CD24 Pos and CD44 + CD24 Neg cell populations by flow cytometry (FACS) using CD44-FITC (mesenchymal marker) and CD24-PE (epithelial marker) monoclonal antibodies (lower plots). The upper plots indicated the blanc (n=3). ( D ) CD24 and CD44 expression was determined by quantitative reverse transcription PCR; the values were normalized to glyceraldehyde 3-phosphate dehydrogenase and relative to control cells. Error bars represent SEM. (*** P <0.001) (n=2). Abbreviations: FACS, fluorescence-activated cell sorting; Neg, negative; Pos, positive.

Article Snippet: Cells were stained with fluorescein isothiocyanate-conjugated mouse anti-human CD44, R-Phycoerythrin-conjugated mouse anti-human CD24 (both from Invitrogen), APC-conjugated mouse anti-human EpCAM (CD326) (Miltenyi Biotec), or APC-conjugated mouse anti-human CD105 (Miltenyi Biotec) monoclonal antibodies.

Techniques: Derivative Assay, Gene Expression, Expressing, Marker, Flow Cytometry, Bioprocessing, Reverse Transcription, Control, Fluorescence, FACS

Journal: Cancer Management and Research

Article Title: CD24 expression and stem-associated features define tumor cell heterogeneity and tumorigenic capacities in a model of carcinogenesis

doi: 10.2147/CMAR.S176654

Figure Lengend Snippet: CD44 + CD24 Pos cells exhibit an enhanced EMT phenotype compared to CD44 + CD24 Neg . Notes: ( A ) Representative images of cell cultures showing cells with fibroblastoid morphology in both cell subpopulations; scale bar, 10 µm. ( B ) Representative FACS histograms (left) showing the expression of Ki-67 in CD44 + CD24 Neg and CD44 + CD24 Pos cells (black and gray histogram, respectively). Filled histogram corresponds to blanc and dotted histogram to isotype control. The cell proliferation capacity (right) was determined by automatic counting of cells at the indicated time points; cells were seeded in triplicates, error bars represent SEM (** P <0.01; *** P <0.001) (n=2). The expression of EMT markers (E-cadherin, Keratin 19, Vimentin, Zeb1, Zeb2, and Twist1) was evaluated by ( C ) Western blot or ( D ) qRT-PCR; the values were normalized to beta-actin or glyceraldehyde 3-phosphate dehydrogenase, respectively (* P <0.05). ( E ) Expression levels of the miR-200 family, relative to the mean expression of hsa-Let7 family (housekeeping gene) as determined by qRT-PCR. Error bars indicate SEM from two independent RT reactions. Abbreviations: EMT, epithelial–mesenchymal transition; Neg, negative; Pos, positive; E-cadh, E-cadherin; Vim, Vimentin; Zeb-1, Zinc Finger E-Box Binding Homeobox 1; Twist-1, Twist Family BHLH Transcription Factor 1.

Article Snippet: Cells were stained with fluorescein isothiocyanate-conjugated mouse anti-human CD44, R-Phycoerythrin-conjugated mouse anti-human CD24 (both from Invitrogen), APC-conjugated mouse anti-human EpCAM (CD326) (Miltenyi Biotec), or APC-conjugated mouse anti-human CD105 (Miltenyi Biotec) monoclonal antibodies.

Techniques: Expressing, Control, Western Blot, Quantitative RT-PCR, Binding Assay

Journal: Cancer Management and Research

Article Title: CD24 expression and stem-associated features define tumor cell heterogeneity and tumorigenic capacities in a model of carcinogenesis

doi: 10.2147/CMAR.S176654

Figure Lengend Snippet: CD44 + CD24 Pos exhibited more ability to migrate and invade but less ability to adhere to CD44 + CD24 Neg cells. Notes: ( A ) Wound healing assay performed in CD44 + CD24 Neg and CD44 + CD24 Pos cells; a representative experiment is shown (micrographs [10×] were taken at the indicated times). Black dotted lines indicate the area of the wound. The histograms show the healed wound area at the different time points; at least ten images were analyzed using the Image J program. Error bars indicate SEM. (* P <0.05; ** P <0.01). ( B ) Transwell migration and ( C ) invasion (toward 20% FBS) assays. Representative micrographs (20×) were taken from the membrane filters (bottom surface of the filters) stained with Crystal violet. The histograms show the quantification of transwell migration and invasion assay by counting the number of cells present on the bottom surface of filters from at least 15 images. Error bars indicate SEM (** P <0.01; *** P <0.001) (n=2). ( D ) Adhesion assay of CD44 + CD24 Neg and CD44 + CD24 Pos cell subpopulations. Representative micrographs (20×) were taken from the plates stained with Crystal violet. The histograms show the quantification of the adhesion assay by counting the number of adherent cells (upper) and by measuring the Crystal violet absorbance (bottom). Error bars indicate SEM (*** P <0.001). Abbreviations: Neg, negative; Pos, positive.

Article Snippet: Cells were stained with fluorescein isothiocyanate-conjugated mouse anti-human CD44, R-Phycoerythrin-conjugated mouse anti-human CD24 (both from Invitrogen), APC-conjugated mouse anti-human EpCAM (CD326) (Miltenyi Biotec), or APC-conjugated mouse anti-human CD105 (Miltenyi Biotec) monoclonal antibodies.

Techniques: Wound Healing Assay, Migration, Membrane, Staining, Invasion Assay, Cell Adhesion Assay

Journal: Cancer Management and Research

Article Title: CD24 expression and stem-associated features define tumor cell heterogeneity and tumorigenic capacities in a model of carcinogenesis

doi: 10.2147/CMAR.S176654

Figure Lengend Snippet: CD44 + CD24 Neg and CD44 + CD24 Pos cell subpopulations differ in their tumorigenic potential. Notes: ( A ) Tumorigenic assay: The indicated sorted cell lines were injected (5×10 5 cells per inoculation) subcutaneously in the flanks of SCID mice. A cell suspension containing 2.5×10 5 cells of each cell subpopulations was used (labeled Mix). As a control, PC1-Expl-1 cells (5×10 5 ) were injected. CD44 + CD24 Neg cells formed visible tumors in 40 days. Error bars indicate mean ± SD. ( B ) Histopathology analyses of tumors formed by PC1-Expl-1, Mix, and CD44 + CD24 Neg cells. H&E staining shows the morphologic aspect. Immunostaining for the epithelial (EMA and E-cadherin) and mesenchymal (αSMA and Vimentin) markers, and for the renal carcinoma marker Pax8, were performed using specific antibodies to detect the human proteins. Abbreviations: αSMA, alpha-smooth muscle actin; EMA, epithelial membrane antigen; Neg, negative; Pos, positive.

Article Snippet: Cells were stained with fluorescein isothiocyanate-conjugated mouse anti-human CD44, R-Phycoerythrin-conjugated mouse anti-human CD24 (both from Invitrogen), APC-conjugated mouse anti-human EpCAM (CD326) (Miltenyi Biotec), or APC-conjugated mouse anti-human CD105 (Miltenyi Biotec) monoclonal antibodies.

Techniques: Injection, Suspension, Labeling, Control, Histopathology, Staining, Immunostaining, Marker, Membrane

Journal: Cancer Management and Research

Article Title: CD24 expression and stem-associated features define tumor cell heterogeneity and tumorigenic capacities in a model of carcinogenesis

doi: 10.2147/CMAR.S176654

Figure Lengend Snippet: CD44 + CD24 Neg and CD44 + CD24 Pos subpopulations vary in their stemness properties. Notes: ( A ) Sphere formation assay in the presence of defined medium (epidermal growth factor and fibroblast growth factor). Representative micrographs (10×) show sphere formation after 8 days of induction. Scale bar, 100 µm. The total number of spheres per well larger than 100 µm was determined at day 8. Error bars indicate SEM. (*** P <0.001) (n=2). ( B ) The gene expression level of stem cell-associated markers (ABC-B1) and ( C ) reprogramming-associated transcription factors was determined by quantitative reverse transcription PCR. The values were normalized to glyceraldehyde 3-phosphate dehydrogenase. Error bars represent SEM (* P <0.05; *** P <0.001) (n=2). ( D ) Representative micrographs of two independent clonogenic experiments in CD44 + CD24 Neg and CD44 + CD24 Pos cells. Colonies were stained with Crystal violet. Abbreviations: Neg, negative; Pos, positive.

Article Snippet: Cells were stained with fluorescein isothiocyanate-conjugated mouse anti-human CD44, R-Phycoerythrin-conjugated mouse anti-human CD24 (both from Invitrogen), APC-conjugated mouse anti-human EpCAM (CD326) (Miltenyi Biotec), or APC-conjugated mouse anti-human CD105 (Miltenyi Biotec) monoclonal antibodies.

Techniques: Tube Formation Assay, Gene Expression, Reverse Transcription, Staining

Journal: Cancer Management and Research

Article Title: CD24 expression and stem-associated features define tumor cell heterogeneity and tumorigenic capacities in a model of carcinogenesis

doi: 10.2147/CMAR.S176654

Figure Lengend Snippet: CD44 + CD24 Neg and CD44 + CD24 Pos subpopulations differ in the expression of stem cell markers. Notes: CD44 + CD24 Pos express high levels of stem cell markers. ( A ) Representative plot showing the expression of EpCAM in CD44 + CD24 Neg and CD44 + CD24 Pos cells (black and gray histogram, respectively) determined by flow cytometry. Dotted histogram corresponds to blanc (n=3). ( B ) ALDH activity was determined by fluorescence-activated cell sorting. DEAB, an ALDH inhibitor, was added to ensure accurate identification of ALDH+ and ALDH− cells. The expression of ( C ) CD133 and ( D ) CD105 was determined by FACS. The histograms show the median fluorescence intensity (n=2). Error bars indicate SEM (* P <0.05; ** P <0.01). Abbreviations: ALDH, aldehyde dehydrogenase; DEAB, diethylamino-benzaldehyde; Neg, negative; Pos, positive.

Article Snippet: Cells were stained with fluorescein isothiocyanate-conjugated mouse anti-human CD44, R-Phycoerythrin-conjugated mouse anti-human CD24 (both from Invitrogen), APC-conjugated mouse anti-human EpCAM (CD326) (Miltenyi Biotec), or APC-conjugated mouse anti-human CD105 (Miltenyi Biotec) monoclonal antibodies.

Techniques: Expressing, Flow Cytometry, Activity Assay, Fluorescence, FACS

Journal: Cancer Management and Research

Article Title: CD24 expression and stem-associated features define tumor cell heterogeneity and tumorigenic capacities in a model of carcinogenesis

doi: 10.2147/CMAR.S176654

Figure Lengend Snippet: CD44 + CD24 Neg and CD44 + CD24 Pos cells differ slightly in their multilineage differentiation capacity. Notes: The differentiation to mesenchymal cell lineages was induced by using specific stimulation media, as described in methods and evaluated after 9 days of induction. Adipocytes (oil red-O), osteoblasts (ALP activity), and chondrocytes (Safranin O) differentiation was determined with specific staining. Representative images are shown. Scale bar, 10 µm. Abbreviations: ALP, alkaline phosphatase; Neg, negative; Pos, positive.

Article Snippet: Cells were stained with fluorescein isothiocyanate-conjugated mouse anti-human CD44, R-Phycoerythrin-conjugated mouse anti-human CD24 (both from Invitrogen), APC-conjugated mouse anti-human EpCAM (CD326) (Miltenyi Biotec), or APC-conjugated mouse anti-human CD105 (Miltenyi Biotec) monoclonal antibodies.

Techniques: Activity Assay, Staining

Journal: Cancer Management and Research

Article Title: CD24 expression and stem-associated features define tumor cell heterogeneity and tumorigenic capacities in a model of carcinogenesis

doi: 10.2147/CMAR.S176654

Figure Lengend Snippet: CD44 + CD24 Pos cells show a proinflammatory gene expression signature. Notes: ( A ) The proinflammatory cytokines IL6 and IL8 were determined using a Becton Dickinson Cytometric Bead Array flow cytometric assay, using serum-free conditioned medium obtained from CD44 + CD24 Neg and CD44 + CD24 Pos cells. Bar graph shows the amount of cytokines in a representative experiment. ( B ) Human proinflammatory cytokines and chemokines were determined using a human cytokine detection kit and protocol (# ARY005B, R&D Systems). Culture media from CD44 + CD24 Neg and CD44 + CD24 Pos cells were blotted on cytokine membranes. Captured proteins were visualized using chemiluminescent detection reagents and the signal produced was quantified. The signal is proportional to the amount of analyte bound. Positive cytokines appear as black dots, in duplicate. + : membrane positive control. *** P <0.001; * P <0.05. Abbreviations: Neg, negative; Pos, positive.

Article Snippet: Cells were stained with fluorescein isothiocyanate-conjugated mouse anti-human CD44, R-Phycoerythrin-conjugated mouse anti-human CD24 (both from Invitrogen), APC-conjugated mouse anti-human EpCAM (CD326) (Miltenyi Biotec), or APC-conjugated mouse anti-human CD105 (Miltenyi Biotec) monoclonal antibodies.

Techniques: Gene Expression, Flow Cytometry, Produced, Membrane, Positive Control

Journal: Cancer Management and Research

Article Title: CD24 expression and stem-associated features define tumor cell heterogeneity and tumorigenic capacities in a model of carcinogenesis

doi: 10.2147/CMAR.S176654

Figure Lengend Snippet: CD44 + CD24 Pos cells showed higher phosphorylation of NF-κB but no changes in the expression or phosphorylation levels of Stat3. Notes: Western blot analysis was performed to assess total and phosphorylated ( A ) NF-κB and ( B ) Stat3 protein levels in CD44 + CD24 Pos and CD44 + CD24 Neg cells. Forty micrograms of proteins were fractionated on SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes and incubated with specific antibodies. ( C ) Quantification of the total (left) and phosphorylated (right) target protein bands relative to β-actin is shown in the bar graphs. Error bars indicate SEM (* P <0.05; ** P <0.01). Abbreviation: NF-κB, nuclear factor kappa B; Neg, negative; Pos, positive.

Article Snippet: Cells were stained with fluorescein isothiocyanate-conjugated mouse anti-human CD44, R-Phycoerythrin-conjugated mouse anti-human CD24 (both from Invitrogen), APC-conjugated mouse anti-human EpCAM (CD326) (Miltenyi Biotec), or APC-conjugated mouse anti-human CD105 (Miltenyi Biotec) monoclonal antibodies.

Techniques: Phospho-proteomics, Expressing, Western Blot, Incubation

Journal: Nucleic Acids Research

Article Title: TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution

doi: 10.1093/nar/gkab642

Figure Lengend Snippet: TDRD3 interacts with DHX9. ( A ) Tandem affinity purification of the TOP3B protein complex (TOP3B.com) from HEK293 cells. The eluted protein complex was separated by SDS-PAGE and silver-stained (left panel). The number of unique peptides from the top four purified proteins is shown (right panel). ( B ) The interaction of DHX9 with TDRD3 and TOP3B was confirmed by co-immunoprecipitation (co-IP). MCF7 cells were immunoprecipitated with control IgG and αTOP3B antibodies. The eluted protein samples were detected by western blot analysis using αDHX9, αTDRD3 and αTOP3B antibodies. ( C ) The interaction of DHX9 with TDRD3 and TOP3B was detected by co-IP in wild type (WT) and TDRD3 knockout (KO) MCF7 cells using two different αTDRD3 antibodies (Ab1 and Ab2). TDRD3, TOP3B and DHX9 were detected in the input samples by western blot analysis. ( D ) TOP3B is dispensable for the TDRD3-DHX9 interaction. Both WT and TOP3B KO MCF7 cells were transfected with Flag-TDRD3. The interaction of TDRD3 with DHX9 and TOP3B was detected by co-IP. ( E ) TDRD3 is essential for the TOP3B-DHX9 interaction. Both WT and TDRD3 KO MCF7 cells were transfected with Flag-TOP3B. The interaction of TOP3B with DHX9 and TDRD3 was detected by co-IP. ( F ) TDRD3 directly interacts with DHX9. Flag-DHX9 recombinant proteins were purified from HEK293 cells. GST, GST-TDRD3, and GST-TOP3B recombinant proteins were purified from E. coli . The interactions of GST-tagged recombinant proteins with Flag-DHX9 were assessed by GST pull-down and western blot analysis using an αFlag antibody. The amount of proteins used in the binding was visualized by the Ponceau S staining of the PVDF membrane.

Article Snippet: Anti-DHX9 rabbit polyclonal antibody (A300-854A) was purchased from Bethyl Laboratories.

Techniques: Affinity Purification, SDS Page, Staining, Purification, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Knock-Out, Transfection, Recombinant, Binding Assay

Journal: Nucleic Acids Research

Article Title: TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution

doi: 10.1093/nar/gkab642

Figure Lengend Snippet: Characterization of the interaction between DHX9 and TDRD3. ( A ) Mapping the region of TDRD3 that interacts with DHX9. GST-tagged TDRD3 truncation constructs were generated, containing the oligonucleotide/oligosaccharide-binding (OB)-fold, the ubiquitin-associated domain (UBA; wild type or with L324A mutation), and the Tudor domain (Tudor; wild type or with E691K mutation), respectively. A graphic summary of their interactions with DHX9 is shown. A GST pull-down assay was performed by incubating the recombinant GST-fusion proteins with MCF7 cell lysates. The pull-down samples were detected by western blot analysis using an αDHX9 antibody. The GST-fusion proteins were visualized by Ponceau staining. ( B ) Mapping the region of DHX9 that interacts with TDRD3. GFP-tagged full-length or truncations of DHX9 constructs were generated. The locations of the N-terminal double-stranded RNA binding domain (dsRBD), the helicase domain (HD), and the C-terminal RGG-containing domain (RGG) are indicated (upper panel). A graphic summary of their interactions with TDRD3 is shown. A GST pull-down assay was performed by incubating the recombinant GST-Tudor with MCF7 lysates that were transfected with different GFP-DHX9 fusion vectors. Both the input and pull-down samples were detected by western blot analysis using an αGFP antibody. The GST-Tudor recombinant protein used in the binding was visualized by Ponceau staining. ( C ) The interaction of TDRD3 with DHX9 requires a functional Tudor domain. A co-IP assay was performed to assess the interaction of Flag-tagged WT and methylarginine binding-deficient (E691K) TDRD3 with endogenous DHX9 in MCF7 cells. The input and αFlag antibody-immunoprecipitated samples were analyzed by western blot using indicated antibodies. ( D ) DHX9 interacts with TDRD3 in an arginine methylation-dependent manner. The interaction of DHX9 with TDRD3 was detected by co-IP in MCF7 cells treated with vehicle (−) or with the type I PRMT inhibitor MS023 (+). The level of cellular ADMA was detected by western blot using a pan-ADMA antibody (ASYM26). The DHX9-immunoprecipitated samples were probed for methylation and interactions with TDRD3 using the ASYM26 and αTDRD3 antibodies.

Article Snippet: Anti-DHX9 rabbit polyclonal antibody (A300-854A) was purchased from Bethyl Laboratories.

Techniques: Construct, Generated, Binding Assay, Mutagenesis, Pull Down Assay, Recombinant, Western Blot, Staining, RNA Binding Assay, Transfection, Functional Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, Methylation

Journal: Nucleic Acids Research

Article Title: TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution

doi: 10.1093/nar/gkab642

Figure Lengend Snippet: TDRD3 recruits DHX9 to its target gene promoters. ( A ) A pie-chart demonstration of genes with TDRD3 bound to their promoters and genes up- or down-regulated after TDRD3 knockout (KO) in MCF7 cells. ( B ) Genes showing more than a 2-fold reduction in TDRD3 KO versus wild type (WT) MCF7 cells were selected for further analysis. The relative expression of each gene in both KO clones is shown. ( C ) UCSC Genome Browser plots of TDRD3 ChIP-seq reads along the indicated genes in MCF7 cells. The y-axis represents the normalized number of reads; the thick blue boxes represent the open reading frames; and the transcription start site (TSS) is labeled. ( D ) A ChIP-reChIP assay was performed to detect the co-occupancy of TDRD3 and DHX9 at target gene promoters. The first round of ChIP was performed using control IgG and αTDRD3 antibodies, and the second round of ChIP (reChIP) was performed using IgG and αDHX9 antibodies. ChIP DNA was analyzed by qPCR using primers for the indicated gene promoters. ( E ) TDRD3 KO does not affect DHX9 protein expression. TDRD3 and DHX9 were detected in WT and TDRD3 KO MCF7 cells by western blot analysis. ACTIN was used as a loading control. ( F ) Loss of TDRD3 reduces DHX9 recruitment to target gene promoters. ChIP assays were performed in WT and TDRD3 KO MCF7 cells using control IgG, αTDRD3, and αDHX9 antibodies. ( G ) Rescued expression of Flag-tagged WT and methylarginine binding-deficient (E691K) TDRD3 in TDRD3 KO MCF7 cells, detected by western blot analysis. ( H ) The methylarginine binding function of TDRD3 is essential for promoting DHX9 recruitment to the target gene promoters. DHX9 ChIP assays were performed in TDRD3 KO MCF7 cells and TDRD3 KO MCF7 cells re-expressing Flag-TDRD3 (WT) and Flag-TDRD3 (E691K). The relative enrichment of DHX9 in TDRD3 KO MCF7 cells was used for normalization. ( I ) DHX9 knockdown reduces TDRD3 target gene expression. MCF7 cells were transfected with either control siRNA (siControl) or DHX9-specific siRNA (siDHX9). The expression of TDRD3 target genes was detected by RT-qPCR assays. Experiments were performed independently for three times. Data are represented as mean ± standard deviation of three technical qPCR replicates. Statistical analysis was performed using Student's t -tests. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Anti-DHX9 rabbit polyclonal antibody (A300-854A) was purchased from Bethyl Laboratories.

Techniques: Knock-Out, Expressing, Clone Assay, ChIP-sequencing, Labeling, Western Blot, Binding Assay, Transfection, Quantitative RT-PCR, Standard Deviation

Journal: Nucleic Acids Research

Article Title: TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution

doi: 10.1093/nar/gkab642

Figure Lengend Snippet: The TDRD3-DHX9 protein complex resolves promoter-associated R-loops. ( A ) Venn diagram of TDRD3 target genes and genes that form R-loops at their promoters, as identified by DRIPc-Seq . ( B ) TDRD3 knockout (KO) increases the R-loop levels at the promoters of its target genes. DRIP-qPCR analysis was performed to compare R-loop levels at the promoters of the TDRD3 target genes in wild type (WT) and TDRD3 KO MCF7 cells. Samples treated with RNase H, which disrupts R-loops, served as negative controls. ( C ) DHX9 knockdown does not affect TDRD3 protein expression. DHX9 and TDRD3 were detected by western blot analysis in MCF7 cells transfected with control siRNA (siControl) or DHX9-specific siRNA (siDHX9). ACTIN was used as a loading control. ( D ) DHX9 knockdown increases R-loop levels at the promoters of TDRD3 target genes. DRIP-qPCR analysis was performed to compare R-loop levels at the promoters of the TDRD3 target genes in control and DHX9 knockdown MCF7 cells. Samples treated with RNase H served as negative controls. ( E ) MCF7 cells were transfected with control siRNA (siControl) or DHX9-specific siRNA (siDHX9). After 24 h, the siDHX9-transfected cells were transfected with either an empty vector, WT DHX9 or helicase activity-deficient (K417R) DHX9 for an additional 48 h. DHX9 and TDRD3 were detected in these cells by western blot analysis. The anti-ACTIN was used as a loading control. ( F ) The helicase activity of DHX9 is essential for resolving R-loops at TDRD3 target gene promoters. DRIP-qPCR analysis was performed to compare R-loop levels at the promoters of the TDRD3 target genes in control (siControl) or DHX9 knockdown (siDHX9) MCF7 cells or DHX9 knockdown MCF7 cells with re-expression of either WT DHX9 (siDHX9 + WT) or helicase activity-deficient DHX9 (siDHX9 + K417R), as described in (E). Samples treated with RNase H served as negative controls. ( G ) TDRD3 KO and DHX9 knockdown cause RNAPII accumulation at gene promoters. RNAPII ChIP assays were performed in WT and TDRD3 KO, as well as control and DHX9 knockdown MCF7 cells. Normal rabbit IgG was used as a negative control. ( H ) Impact of TDRD3 KO and DHX9 knockdown on the nascent RNA transcription. Diagram demonstration of RHOB and RAD23A gene locus. The bars at the bottom of the diagrams indicate the amplicon locations for nascent RNA transcripts (red: promoter; blue: gene body and 3′end). Chromatin RNA immunoprecipitation was performed in WT and TDRD3 KO, as well as control and DHX9 knockdown MCF7 cells. The levels of nascent RNA were quantified by RT-qPCR. Experiments were performed independently three times. Data are represented as mean ± standard deviation of three technical qPCR replicates. Statistical analysis was performed using Student's t -tests. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Anti-DHX9 rabbit polyclonal antibody (A300-854A) was purchased from Bethyl Laboratories.

Techniques: Knock-Out, Expressing, Western Blot, Transfection, Plasmid Preparation, Activity Assay, Negative Control, Amplification, Immunoprecipitation, Quantitative RT-PCR, Standard Deviation

Journal: Nucleic Acids Research

Article Title: TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution

doi: 10.1093/nar/gkab642

Figure Lengend Snippet: TDRD3 promotes DHX9 helicase activity in R-loop resolution. ( A ) DHX9 resolves R-loops in a helicase activity-dependent manner. Coomassie Blue staining of recombinant wild type (WT) and helicase activity-deficient (K417R) DHX9 purified from HEK293 cells (left). The helicase assay on R-loops was performed by incubating increasing amounts of recombinant WT or K417R DHX9 with 5′ 6-FAM-labeled R-loop substrates (5 nM) at 37°C for 10 min. The reaction products were analyzed by gel electrophoresis and fluorescence imaging (right). The open triangle indicates the recombinant proteins. ( B ) TDRD3 stimulates the helicase activity of WT DHX9, but not K417R mutant DHX9, in R-loop resolution. Coomassie Blue staining of recombinant WT, K417R mutant DHX9, and TDRD3 purified from HEK293 cells (left). The helicase assay on R-loops was performed by incubating 5′ 6-FAM-labeled R-loop substrates with constant amounts of either WT or K417R mutant DHX9 (2 nM) and increasing amounts (40 and 60 nM) of TDRD3 (right). The open triangles indicate the recombinant proteins. ( C ) The OB-fold of TDRD3 binds single-stranded DNA (ssDNA). A graphic summary of the interactions of truncated TDRD3 fragments with ssDNA is shown (upper panel). Coomassie Blue staining of recombinant GST-fusion proteins of TDRD3, including its OB-fold, UBA domain, and Tudor domain, purified from E. coli (lower left panel). The electrophoretic mobility shift assay (EMSA) was performed by incubating a 5′ 6-FAM-labeled ssDNA oligonucleotide (5 nM) with increasing amounts (25 and 50 nM) of the recombinant TDRD3 proteins (lower right panel). The solid triangle indicates the protein–nucleotide complex. ( D ) The OB-fold is essential for the interaction of TDRD3 with ssDNA. A graphic summary of the WT, methylarginine binding-deficient (E691K), and OB-fold-truncated (ΔOB) TDRD3 interaction with ssDNA is shown (upper panel). Coomassie Blue staining of all three recombinant TDRD3 proteins purified from HEK293 cells (lower left panel). EMSA was performed to detect the binding of increasing amounts (20, 40 and 60 nM) of recombinant proteins with a 5′ 6-FAM-labeled ssDNA oligonucleotide (5 nM) (lower right panel). The open triangle indicates the recombinant proteins. The solid triangle indicates the protein-nucleotide complex. ( E ) TDRD3 interacts with R-loops, but not DNA/RNA hybrids. EMSA was performed by incubating increasing amounts (40 and 60 nM) of recombinant WT and OB-fold-truncated (ΔOB) TDRD3 with 5′ 6-FAM-labeled R-loop or DNA/RNA hybrid oligonucleotide (5 nM). The open triangle indicates the recombinant proteins. The solid triangle indicates the protein-nucleotide complex. ( F ) Both the OB-fold and the functional Tudor domain are required for TDRD3 to stimulate the helicase activity of DHX9 in R-loop resolution. The helicase assay on R-loops was performed by incubating 5′ 6-FAM-labeled R-loop substrates (5 nM) with a constant amount of DHX9 (2 nM) and increasing amounts (20, 40 and 60 nM) of WT, methylarginine binding-deficient (E691K), and ssDNA binding-deficient (ΔOB) TDRD3 (left). Helicase activity was quantified by measuring the percentage of unwound substrates under the indicated assay conditions (right). Statistical analysis was performed using Student's t -tests of data from three independent experiments. *** P < 0.001.

Article Snippet: Anti-DHX9 rabbit polyclonal antibody (A300-854A) was purchased from Bethyl Laboratories.

Techniques: Activity Assay, Staining, Recombinant, Purification, Helicase Assay, Labeling, Nucleic Acid Electrophoresis, Fluorescence, Imaging, Mutagenesis, Electrophoretic Mobility Shift Assay, Binding Assay, Functional Assay

Journal: Nucleic Acids Research

Article Title: TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution

doi: 10.1093/nar/gkab642

Figure Lengend Snippet: DHX9 and TOP3B function together to resolve co-transcriptional R-loops. ( A ) A ChIP-reChIP assay was performed to detect the co-occupancy of TDRD3 and TOP3B at target gene promoters. The first round of ChIP was performed using control IgG and αTDRD3 antibodies, and the second round of ChIP (reChIP) was performed using IgG and αTOP3B antibodies. ChIP DNA was analyzed by qPCR using primers for the indicated gene promoters. ( B ) Loss of TDRD3 reduces TOP3B recruitment to target gene promoters. ChIP assays were performed in WT and TDRD3 KO MCF7 cells using control IgG, αTDRD3, and αTOP3B antibodies. ( C ) MCF7 cells were transfected with control siRNA (siControl), DHX9-specific siRNA (siDHX9), or TOP3B-specific siRNA (siTOP3B), individually or in combination. TOP3B, DHX9 and TDRD3 were detected by western blot analysis using indicated antibodies. ( D ) TOP3B and DHX9 function together in resolving R-loops at TDRD3 target gene promoters. DRIP-qPCR analysis was performed to compare R-loop levels at the promoters of TDRD3 target genes in control (siControl), DHX9 knockdown (siDHX9), TOP3B knockdown (siTOP3B), and DHX9/TOP3B double knockdown (siDHX9 + siTOP3B) MCF7 cells. Samples treated with RNase H served as negative controls. ( E ) Schematic of the in vitro plasmid-based R-loop formation assay followed by quantification of R-loop levels using Dot-blot and DRIP-qPCR. pFC53 plasmids, which contain the R-loop-forming sequence of Airn (13), were transcribed in vitro using T3 RNA polymerase under standard conditions. The transcription products, including the DNA template, the free RNA product, and R-loop containing template, were then subjected to RNase A digestion to remove RNA. The remaining samples were equally divided and either left untreated (−) or treated with RNase H (+). The final products were purified and subjected to either Dot-blot analysis or DRIP-qPCR with DNA/RNA hybrid-specific antibody (S9.6) for R-loop quantification. ( F ) TOP3B and DHX9 function together to resolve co-transcriptional R-loops in vitro . The pFC53 plasmid was subjected to in vitro transcription in the presence of DHX9, WT TOP3B, and topoisomerase activity-deficient (Y336F) TOP3B, either individually or in combination, as indicated. The level of R-loops in each reaction was detected by Dot-Blot using the S9.6 antibody. The loading of the nucleic acid was visualized by methylene blue staining of the membrane. ( G ) TDRD3 functions as a scaffold that assembles a protein complex, containing TOP3B and DHX9, to regulate co-transcriptional R-loops at gene promoters. TDRD3 not only recruits TOP3B and DHX9 to specific genomic regions, but also stimulates their respective enzymatic activities to either resolve underwound DNA to prevent R-loop formation (with TOP3B) or resolve existing R-loops to avoid R-loop accumulation (with DHX9). This activity of TDRD3 is likely conferred by its interaction with ssDNA.

Article Snippet: Anti-DHX9 rabbit polyclonal antibody (A300-854A) was purchased from Bethyl Laboratories.

Techniques: Transfection, Western Blot, In Vitro, Plasmid Preparation, Tube Formation Assay, Dot Blot, Sequencing, Purification, Activity Assay, Staining

Journal: Nature

Article Title: Targeted protein degradation via intramolecular bivalent glues

doi: 10.1038/s41586-024-07089-6

Figure Lengend Snippet: a , BRD4 stability CRISPR screen. KBM7 iCas9 BRD4 dual fluorescence reporter cells expressing a CRL-focused sgRNA library were treated with GNE-0011 (1 µM) for 6 h before flow cytometric cell sorting into BRD4 low , BRD4 mid and BRD4 high fractions as in Fig. . 20 S proteasome subunits (blue), COP9 signalosome subunits (cyan) and E1 or E2 ubiquitin enzymes (purple) inside the scoring window (one-sided MAGeCK p-value < 0.01, fold-change > 1.5; dashed lines) are highlighted. b – f , Immunoblot-based CRISPR/Cas9 screen validation. b , CRISPR-based validation. KBM7 iCas9 cells were lentivirally transduced with sgRNAs targeting AAVS1, DCAF16 or DDB1 and 3 days after Cas9 induction, cells were treated with GNE-0011 (1 µM), dBET6 (10 nM) or IBG1 (1 nM) for 6 h and BRD4 levels were analysed via immunoblot. Data are representative of n = 2 independent experiments. c – e , siRNA-based validation. HCT-116 cells were transfected with siRNA pools targeting the indicated genes and treated with DMSO, IBG1, GNE-0011 or dBET6 for 2 h at the indicated concentrations and BET protein levels were analysed via immunoblotting. Data are representative of n = 2 independent experiments. f , DCAF16 knockout/rescue. KBM7 iCas9 cells were lentivirally transduced with DCAF16 -targeting or AAVS1 control sgRNAs, as well as a DCAF16 cDNA in which the sgRNA target sites were removed by synonymous mutations. After knockout of endogenous DCAF16 and compound treatment for 6 h as above, BRD4 expression levels were assessed via immunoblotting (n = 1). g , Induction of apoptosis. KBM7 iCas9 WT or DCAF16 knockout cells were treated with increasing concentrations of IBG1, GNE-0011 or dBET6 for 16 h and levels of BRD4, MYC, cleaved PARP1 and cleaved caspase 3 were analysed via immunoblotting as in Fig. . Data are representative of n = 3 independent experiments.

Article Snippet: The following primary antibodies were used: BRD2 (1:1,000, no. Ab139690, Abcam), BRD3 (1:2,000, Ab50818, Abcam), BRD4 (1:1,000, E2A7X, 13440, Cell Signaling Technology and Ab128874, Abcam), BromoTag (1:1,000, NBP3-17999, Novus Biologicals), CUL4A (1:2,000, A300-738A, Bethyl Laboratories), CUL4B (1:2,000, 12916-1-AP, Proteintech), DDB1 (1:1,000, A300-462A, Bethyl Laboratories), MCM4 (1:1,000, ab4459, Abcam) RBM39 (1:1,000, HPA001591, Atlas Antibodies), RBX1 (1:1,000, D3J5I, 11922, Cell Signalling Technology), DCAF11 (1:2,000, A15519, ABclonal), cleaved caspase-3 (1:1,000, D3E9, 9579, Cell Signalling Technology), PARP1 (1:1,000, 9542, Cell Signalling Technology), MYC (1:500, D84C12, 5605, Cell Signalling Technology), β-actin (1:10,000, AC-15, A5441, Sigma-Aldrich), α-tubulin (1:500, DM1A, T9026, Sigma-Aldrich).

Techniques: CRISPR, Fluorescence, Expressing, FACS, Western Blot, Transduction, Transfection, Knock-Out

Journal: Nature

Article Title: Targeted protein degradation via intramolecular bivalent glues

doi: 10.1038/s41586-024-07089-6

Figure Lengend Snippet: a , Schematic of FACS-based CRISPR–Cas9 screens. Doxycycline (Dox)-inducible Cas9 (iCas9) KBM7 BRD4–BFP reporter cells were transduced with a CRL-focused sgRNA library, treated with BET degraders and sorted based on BRD4–BFP/mCherry ratios. b , FACS-based CRISPR screens for BRD4 stability. KBM7 iCas9 BRD4 reporter cells were treated with DMSO, MZ1 (10 nM) or IBG1 (1 nM) for 6 h before sorting. 20S proteasome subunits, COP9 signalosome subunits and E1 and E2 ubiquitin enzymes inside the scoring window (one-sided MAGeCK P value < 0.01, fold change > 1.5) are highlighted. c , CRISPR–Cas9 viability screen. HCT-116 cells were transduced with Cas9 and a ubiquitin–proteasome system-focused sgRNA library and treated with IBG1 (58 nM; fourfold half-maximal inhibitory concentration (IC 50 )) for 6 days. Genes with a fold change > 2 and one-sided MAGeCK P value < 0.01 are highlighted. d , Screen validation. KBM7 iCas9 BRD4–BFP reporter cells were transduced with AAVS1 , DCAF16 or DDB1 -targeting sgRNAs, treated with DMSO, IBG1 (1 nM) or dBET6 (10 nM) for 6 h, and BRD4–BFP was quantified by FACS. e , DCAF16 knockout and rescue. KBM7 iCas9 BRD4–BFP reporter cells were transduced with AAVS1 or DCAF16 -targeting sgRNAs, with or without sgRNA-resistant DCAF16 cDNA. After knockout of endogenous DCAF16, cells were treated for 6 h as in b and BRD4–BFP was quantified by FACS. f , Apoptosis induction. Wild-type or DCAF16 -knockout KBM7 cells were treated with indicated concentrations of dBET6 or IBG1 for 16 h. Cleaved PARP1 was evaluated by immunoblotting. g , Viability assay. Wild-type or DCAF16 -knockout KBM7 cells were treated with IBG1 or dBET6 for 72 h and cell viability was evaluated by CellTiterGlo assay. Mean ± s.d. of n = 3 biological replicates. h , Fluorescence polarization binary binding assay. FITC-labelled sulfonamide probe was titrated into DCAF15–DDB1(ΔBPB)–DDA1 or DCAF16–DDB1(ΔBPB)–DDA1. DDB1(ΔBPB) lacks the cullin-binding domain (BPB). n = 3 technical replicates. d – f , n = 3 independent experiments. d – h , Mean ± s.d.

Article Snippet: The following primary antibodies were used: BRD2 (1:1,000, no. Ab139690, Abcam), BRD3 (1:2,000, Ab50818, Abcam), BRD4 (1:1,000, E2A7X, 13440, Cell Signaling Technology and Ab128874, Abcam), BromoTag (1:1,000, NBP3-17999, Novus Biologicals), CUL4A (1:2,000, A300-738A, Bethyl Laboratories), CUL4B (1:2,000, 12916-1-AP, Proteintech), DDB1 (1:1,000, A300-462A, Bethyl Laboratories), MCM4 (1:1,000, ab4459, Abcam) RBM39 (1:1,000, HPA001591, Atlas Antibodies), RBX1 (1:1,000, D3J5I, 11922, Cell Signalling Technology), DCAF11 (1:2,000, A15519, ABclonal), cleaved caspase-3 (1:1,000, D3E9, 9579, Cell Signalling Technology), PARP1 (1:1,000, 9542, Cell Signalling Technology), MYC (1:500, D84C12, 5605, Cell Signalling Technology), β-actin (1:10,000, AC-15, A5441, Sigma-Aldrich), α-tubulin (1:500, DM1A, T9026, Sigma-Aldrich).

Techniques: CRISPR, Transduction, Concentration Assay, Knock-Out, Western Blot, Viability Assay, Fluorescence, Binding Assay

Journal: Nature

Article Title: Targeted protein degradation via intramolecular bivalent glues

doi: 10.1038/s41586-024-07089-6

Figure Lengend Snippet: a , ITC measurement of DCAF16–DDB1(ΔBPB)–DDA1 binding to pre-incubated BRD4 Tandem –IBG1 complex (1:1.1 molar ratio). Data representative of n = 2 independent experiments. DP, differential power. b , TR-FRET ternary complex-formation assay. Europium-labelled anti-His bound to BRD4 Tandem was incubated with equimolar Cy5-labelled DCAF16–DDB1(ΔBPB)–DDA1 and increasing concentrations of IBG1 or JQ1. Mean ± s.d. of n = 3 technical replicates. c , TR-FRET complex-stabilization assay. His-tagged BRD4 Tandem - or BRD4 BD1 (200 nM) bound to anti-His–europium was incubated with increasing concentrations of Cy5-labelled DCAF16–DDB1(ΔBPB)–DDA1 in the presence or absence of 1 µM IBG1. Mean ± s.d. of n = 2 independent experiments, each with 2 technical replicates. The asterisk denotes a datapoint that was excluded from non-linear regression fitting. d , e , UV chromatograms from SEC analysis. DCAF16–DDB1(ΔBPB)–DDA1 and BRD4 Tandem alone or mixed at a 2:1 molar ratio in the presence of excess IBG1 ( d ), DCAF16–DDB1(ΔBPB)–DDA1 and BRD4 Tandem mixed at a 1:1 molar ratio in the absence or presence of excess IBG1 ( e ), or DCAF16–DDB1(ΔBPB)–DDA1 mixed with BRD4 BD1 and BRD4 BD2 at a molar ratio of 1:1:1 with excess IBG1 ( e ) were run on an S200 10/300 column. Data representative of n = 2 independent experiments. mAU, milli-absorbance units. f , BET protein stability reporter assay. Tandem mTagBFP fusions with BRD2, BRD3 or BRD4 bromodomains, isolated BRD4 bromodomains or bromodomain chimeras were expressed in KBM7 cells and protein stability was quantified by FACS following treatment with DMSO, IBG1 (1 nM) or dBET6 (10 nM) for 6 h. Mean ± s.d. of n = 3 independent experiments.

Article Snippet: The following primary antibodies were used: BRD2 (1:1,000, no. Ab139690, Abcam), BRD3 (1:2,000, Ab50818, Abcam), BRD4 (1:1,000, E2A7X, 13440, Cell Signaling Technology and Ab128874, Abcam), BromoTag (1:1,000, NBP3-17999, Novus Biologicals), CUL4A (1:2,000, A300-738A, Bethyl Laboratories), CUL4B (1:2,000, 12916-1-AP, Proteintech), DDB1 (1:1,000, A300-462A, Bethyl Laboratories), MCM4 (1:1,000, ab4459, Abcam) RBM39 (1:1,000, HPA001591, Atlas Antibodies), RBX1 (1:1,000, D3J5I, 11922, Cell Signalling Technology), DCAF11 (1:2,000, A15519, ABclonal), cleaved caspase-3 (1:1,000, D3E9, 9579, Cell Signalling Technology), PARP1 (1:1,000, 9542, Cell Signalling Technology), MYC (1:500, D84C12, 5605, Cell Signalling Technology), β-actin (1:10,000, AC-15, A5441, Sigma-Aldrich), α-tubulin (1:500, DM1A, T9026, Sigma-Aldrich).

Techniques: Binding Assay, Incubation, Tube Formation Assay, Reporter Assay, Isolation

Journal: Nature

Article Title: Targeted protein degradation via intramolecular bivalent glues

doi: 10.1038/s41586-024-07089-6

Figure Lengend Snippet: a , Electron density (left) and model (right) of the complex formed between DCAF16, DDB1(ΔBPB), BRD4 Tandem (BD1 and BD2) and IBG1. b , Electron density at the DCAF16–IBG1–BRD4 interface. The JQ1 moiety binds to BD2, and the sulfonamide engages BD1. c , UV chromatograms from SEC analysis. Recombinant BRD4 Tandem was incubated with DMSO, JQ1 or IBG1 at a 1:2 molar ratio and run on an S200 10/300 column. Data representative of n = 2 independent experiments. d , A hydrophobic cage formed by DCAF16 residues C58, L59, Y62 and W181 encloses the JQ1 moiety and linker phenyl ring of IBG1. e , Selectivity-determining residue G386 of BD2 at the interface with DCAF16. Colours in b, d , e as in a . f , FACS reporter assay. KBM7 reporter cells expressing wild-type BRD3, BRD4 or indicated single-point mutant bromodomain tandems were treated with IBG1 (1 nM) or dBET6 (10 nM) for 6 h and BET protein stability was evaluated by FACS. Mean ± s.d. of n = 3 independent experiments. g – j , Structure ( g ) and mechanistic characterization ( h – j ) of the dual-JQ1-containing BET degrader IBG3. h , BRD4 degradation. KBM7 reporter cells expressing BRD4 Tandem were treated for 6 h with increasing concentrations of IBG1, IBG3 or dBET6, and BRD4 protein stability was assessed by FACS. Mean ± s.d. of n = 3 independent experiments. i , TR-FRET ternary complex-formation assay. Anti-His–europium bound to BRD4 Tandem was incubated with equimolar Cy5-labelled DCAF16–DDB1(ΔBPB)–DDA1 and increasing concentrations of IBG1, IBG3 or JQ1. Data for JQ1 and IBG1 as in Fig. . Mean ± s.d. of n = 3 technical replicates. j , ITC measurements of DCAF16–DDB1(ΔBPB)–DDA1 complex binding to pre-incubated BRD4 Tandem –IBG3 (1:1.1 molar ratio). Data representative of n = 2 independent experiments.

Article Snippet: The following primary antibodies were used: BRD2 (1:1,000, no. Ab139690, Abcam), BRD3 (1:2,000, Ab50818, Abcam), BRD4 (1:1,000, E2A7X, 13440, Cell Signaling Technology and Ab128874, Abcam), BromoTag (1:1,000, NBP3-17999, Novus Biologicals), CUL4A (1:2,000, A300-738A, Bethyl Laboratories), CUL4B (1:2,000, 12916-1-AP, Proteintech), DDB1 (1:1,000, A300-462A, Bethyl Laboratories), MCM4 (1:1,000, ab4459, Abcam) RBM39 (1:1,000, HPA001591, Atlas Antibodies), RBX1 (1:1,000, D3J5I, 11922, Cell Signalling Technology), DCAF11 (1:2,000, A15519, ABclonal), cleaved caspase-3 (1:1,000, D3E9, 9579, Cell Signalling Technology), PARP1 (1:1,000, 9542, Cell Signalling Technology), MYC (1:500, D84C12, 5605, Cell Signalling Technology), β-actin (1:10,000, AC-15, A5441, Sigma-Aldrich), α-tubulin (1:500, DM1A, T9026, Sigma-Aldrich).

Techniques: Recombinant, Incubation, Residue, Reporter Assay, Expressing, Mutagenesis, Tube Formation Assay, Binding Assay

Journal: Nature

Article Title: Targeted protein degradation via intramolecular bivalent glues

doi: 10.1038/s41586-024-07089-6

Figure Lengend Snippet: a , Structure of DCAF16 coloured rainbow from N- to C-terminus. b , Comparison of DCAF16 and structurally distinct CRL4 substrate receptors DCAF1, DCAF15, and CRBN (PDB entries 5JK7, 6UD7, and 5FQD, respectively) bound to DDB1 (blue). c , Comparison of binding mode in the acetyl-lysine pocket of BRD4 BD1 (orange surface and cartoon) between IBG1 (orange) and known sulfonamide BET inhibitors PFI-1 (left, light blue; PDB 4E96) and compound 6j (right, grey; PDB 5Y94). The cyano group of IBG1 overlays close to a conserved water molecule found in both crystal structures and other published BD1 structures. d , Fluorescence polarization binary binding assay. Proteins were titrated into 20 nM FITC-sulfonamide probe, as in Fig. . n = 3 technical replicates, mean +/− s.d. e , alphaLISA displacement assay. Competition of a biotinylated-JQ1 probe following titration of compounds 1a , 1d , E7820, Indisulam, or JQ1 into His-BRD4 BD1 (left) or His-BRD4 BD2 (right). Data, mean of n = 2 technical replicates. f , Detailed view of DCAF16-BD1 interface. Residue W54 of DCAF16 binds to a hydrophobic pocket on the surface of BD1. g , Detail view of BD1-BD2 interface. Residue M442 of BD2 is sandwiched between residues W81 and P375 of the BD1 and BD2 WPF shelves, respectively, as well as the linker of IBG1.

Article Snippet: The following primary antibodies were used: BRD2 (1:1,000, no. Ab139690, Abcam), BRD3 (1:2,000, Ab50818, Abcam), BRD4 (1:1,000, E2A7X, 13440, Cell Signaling Technology and Ab128874, Abcam), BromoTag (1:1,000, NBP3-17999, Novus Biologicals), CUL4A (1:2,000, A300-738A, Bethyl Laboratories), CUL4B (1:2,000, 12916-1-AP, Proteintech), DDB1 (1:1,000, A300-462A, Bethyl Laboratories), MCM4 (1:1,000, ab4459, Abcam) RBM39 (1:1,000, HPA001591, Atlas Antibodies), RBX1 (1:1,000, D3J5I, 11922, Cell Signalling Technology), DCAF11 (1:2,000, A15519, ABclonal), cleaved caspase-3 (1:1,000, D3E9, 9579, Cell Signalling Technology), PARP1 (1:1,000, 9542, Cell Signalling Technology), MYC (1:500, D84C12, 5605, Cell Signalling Technology), β-actin (1:10,000, AC-15, A5441, Sigma-Aldrich), α-tubulin (1:500, DM1A, T9026, Sigma-Aldrich).

Techniques: Comparison, Binding Assay, Fluorescence, Titration, Residue

Journal: Nature

Article Title: Targeted protein degradation via intramolecular bivalent glues

doi: 10.1038/s41586-024-07089-6

Figure Lengend Snippet: a , Structure of IBG4. b , Tandem bromodomain requirement of IBG4. KBM7 reporter cells expressing BRD4 Tandem , BD1 or BD2 were treated for 6 h with increasing concentrations of IBG4, and protein degradation was evaluated by FACS. Mean ± s.d. of n = 3 independent experiments. c , UV chromatograms from SEC analysis of BRD4 Tandem incubated with DMSO, JQ1, IBG1 or IBG4. Data representative of n = 2 independent experiments. Data for DMSO, JQ1 and IBG1 as in Fig. . d , CRISPR–Cas9 BRD4 stability screen. KBM7 iCas9 BRD4 reporter cells expressing a CRL-focused sgRNA library were treated with IBG4 (100 nM) for 6 h before FACS as in Fig. . The 20S proteasome subunits, COP9 signalosome subunits and E1 and E2 ubiquitin enzymes inside the scoring window (one-sided MAGeCK P value < 0.01, fold change > 1.5; dashed lines) are highlighted. e , Immunoblot-based screen validation. AAVS1 control, DCAF11 - or DCAF16 -knockout KBM7 cells were treated with DMSO, IBG4 (100 nM) or IBG3 (0.1 nM) for 6 h and BRD4 was quantified by immunoblotting. The asterisk denotes a nonspecific band. Data representative of n = 3 independent experiments. f , TR-FRET complex-stabilization assay. His-tagged BRD4 Tandem (100 nM) bound to anti-His–europium was incubated with increasing concentrations of Cy5-labelled DCAF11–DDB1(ΔBPB)–DDA1 and 500 nM IBG4 or DMSO. Mean ± s.d. of n = 2 independent experiments, each with 3 technical replicates. g , TR-FRET complex-formation assay. His-tagged BRD4 Tandem bound to anti-His–europium was incubated with equimolar Cy5-labelled DCAF11–DDB1(ΔBPB)–DDA1 and increasing concentrations of IBG4 or JQ1. Mean ± s.d. of n = 3 technical replicates. h , Schematic model of the different modes of molecular recognition with traditional monovalent glues and bivalent PROTACs versus intramolecularly bivalent glues revealed in this work.

Article Snippet: The following primary antibodies were used: BRD2 (1:1,000, no. Ab139690, Abcam), BRD3 (1:2,000, Ab50818, Abcam), BRD4 (1:1,000, E2A7X, 13440, Cell Signaling Technology and Ab128874, Abcam), BromoTag (1:1,000, NBP3-17999, Novus Biologicals), CUL4A (1:2,000, A300-738A, Bethyl Laboratories), CUL4B (1:2,000, 12916-1-AP, Proteintech), DDB1 (1:1,000, A300-462A, Bethyl Laboratories), MCM4 (1:1,000, ab4459, Abcam) RBM39 (1:1,000, HPA001591, Atlas Antibodies), RBX1 (1:1,000, D3J5I, 11922, Cell Signalling Technology), DCAF11 (1:2,000, A15519, ABclonal), cleaved caspase-3 (1:1,000, D3E9, 9579, Cell Signalling Technology), PARP1 (1:1,000, 9542, Cell Signalling Technology), MYC (1:500, D84C12, 5605, Cell Signalling Technology), β-actin (1:10,000, AC-15, A5441, Sigma-Aldrich), α-tubulin (1:500, DM1A, T9026, Sigma-Aldrich).

Techniques: Expressing, Incubation, CRISPR, Western Blot, Knock-Out, Tube Formation Assay

Journal: Nature

Article Title: Targeted protein degradation via intramolecular bivalent glues

doi: 10.1038/s41586-024-07089-6

Figure Lengend Snippet: a , Bromodomain tandem specificity. KBM7 cells expressing bromodomain mutant BRD4 Tandem dual fluorescence reporters were treated with DMSO, IBG4 (100 nM) or dBET6 (10 nM) for 6 h and analysed by flow cytometry. b , NanoBRET bromodomain dimerization assay. Indicated compounds were titrated into HEK293 cells transiently expressing BRD4 Nluc-Tandem-HaloTag . Data, mean of n = 2 independent experiments. c , alphaLISA displacement assay. Increasing concentrations of JQ1, E7820 or the pyrazolo pyrimidine warhead of IBG4 were titrated against His-tagged BRD4 bromodomains and biotinylated JQ1 probe. n = 3 technical replicates, mean +/− s.d. d , BET protein selectivity. Bromodomain tandem BRD2, BRD3 or BRD4 dual fluorescence reporter KBM7 cells were treated with DMSO, IBG4 (100 nM) or dBET6 (10 nM) for 6 h and analysed by flow cytometry. e , Mechanistic FACS reporter assay. KBM7 BRD4 dual fluorescence reporter cells were co-treated with IBG1 (1 nM) or IBG4 (100 nM) and Carfilzomib (1 µM), MLN4924 (1 µM) or TAK243 (0.5 µM) for 6 h and BRD4 levels were analysed via flow cytometry. Data, mean of n = 2 independent experiments. f , DCAF16-independence of IBG4. KBM7 iCas9 WT or DCAF16 knockout cells expressing BRD4(S) dual fluorescence reporter were treated with DMSO, IBG1 (1 nM) or IBG4 (100 nM) for 6 h and BRD4 degradation was assessed via flow cytometry. g , AlphaFold (AlphaFold Monomer v2.0 pipeline) prediction of DCAF11 (red) bound to DDB1 (blue). h , i , Size exclusion chromatograms of different combinations of DCAF11, BRD4 Tandem and IBG4 ( h ), data representative of n = 2 independent experiments, and corresponding peak fractions run on SDS-PAGE ( i ). Data for a , d , f , n = 3 independent experiments, mean +/− s.d.

Article Snippet: The following primary antibodies were used: BRD2 (1:1,000, no. Ab139690, Abcam), BRD3 (1:2,000, Ab50818, Abcam), BRD4 (1:1,000, E2A7X, 13440, Cell Signaling Technology and Ab128874, Abcam), BromoTag (1:1,000, NBP3-17999, Novus Biologicals), CUL4A (1:2,000, A300-738A, Bethyl Laboratories), CUL4B (1:2,000, 12916-1-AP, Proteintech), DDB1 (1:1,000, A300-462A, Bethyl Laboratories), MCM4 (1:1,000, ab4459, Abcam) RBM39 (1:1,000, HPA001591, Atlas Antibodies), RBX1 (1:1,000, D3J5I, 11922, Cell Signalling Technology), DCAF11 (1:2,000, A15519, ABclonal), cleaved caspase-3 (1:1,000, D3E9, 9579, Cell Signalling Technology), PARP1 (1:1,000, 9542, Cell Signalling Technology), MYC (1:500, D84C12, 5605, Cell Signalling Technology), β-actin (1:10,000, AC-15, A5441, Sigma-Aldrich), α-tubulin (1:500, DM1A, T9026, Sigma-Aldrich).

Techniques: Expressing, Mutagenesis, Fluorescence, Flow Cytometry, Reporter Assay, Knock-Out, SDS Page

Journal: International Journal of Molecular Medicine

Article Title: CENPU promotes tumorigenesis and stem cell properties in triple-negative breast cancer by suppressing lysosomal furin degradation

doi: 10.3892/ijmm.2025.5649

Figure Lengend Snippet: CENPU promotes tumorigenesis, stem cell-like properties, migration and invasion in breast cancer cells. Western blotting was performed to detect the expression levels of CD44 and ALDH in (A) MDA-MB-231, (B) Cal51 and (C) T47D cells with different CENPU expression levels. Association between CENPU expression and the number of mammospheres per 200 cells in (E) MDA-MB-231 and Cal51 cells and (G) T47D cells (scale bar, 100 μ m). CD24 − CD44 + flow cytometry results showed that CENPU expression levels affected the percentage of the CD24 − CD44 + population among (D) MDA-MB-231, (F) Cal51 and (H) T47D cells. Transwell assays were performed to determine the effect of CENPU levels on the migration and invasiveness of (I) MDA-MB-231, (J) Cal51 and (K) T47D cells (scale bar, 200 μ m). A wound healing assay was performed to determine the effect of CENPU on the migration potential of (L) MDA-MB-231, (M) Cal51 and (N) T47D cells (scale bar, 200 μ m). * P<0.05; ** P<0.01. CENPU, centromere protein U; sh, short hairpin RNA; APC, allophycocyanin; ALDH, aldehyde dehydrogenase; PE, phycoerythrin.

Article Snippet: Human MDA-MB-231 (TNBC) and T47D (BC) cells were acquired from the American Type Culture Collection.

Techniques: Migration, Western Blot, Expressing, Flow Cytometry, Wound Healing Assay, shRNA

Journal: International Journal of Molecular Medicine

Article Title: CENPU promotes tumorigenesis and stem cell properties in triple-negative breast cancer by suppressing lysosomal furin degradation

doi: 10.3892/ijmm.2025.5649

Figure Lengend Snippet: CENPU promotes the conversion of proNGF to NGF. Western blotting experiments were performed to detect the expression of NGF and proNGF in (A) MDA-MB-231-s hCENPU , (B) Cal51-sh CENPU and (C) T47D- CENPU cells and their corresponding control groups. The intracellular NGF levels in (D) MDA-MB-231, (E) Cal51 and (F) T47D cells after 48 h of treatment were analyzed by ELISA using ultrasonicated cell supernatants. After NGF-neutralizing antibodies were added to (G) MDA-MB-231-s hCENPU , (I) Cal51-sh CENPU and (K) T47D- CENPU cells, and their corresponding control groups, Transwell migration and invasion assays were conducted (scale bar, 200 μ m). After NGF-neutralizing antibodies were added to the (H) MDA-MB-231-s hCENPU , (J) Cal51-sh CENPU and (L) T47D- CENPU cells, and their corresponding control groups, a mammosphere formation assay was performed to measure the number of mammospheres per 200 cells that formed (scale bar, 100 μ m). * P<0.05; ** P<0.01. CENPU, centromere protein U; sh, short hairpin RNA; NGF, nerve growth factor; proNGF, NGF precursor; NS, not significant.

Article Snippet: Human MDA-MB-231 (TNBC) and T47D (BC) cells were acquired from the American Type Culture Collection.

Techniques: Western Blot, Expressing, Control, Enzyme-linked Immunosorbent Assay, Migration, Tube Formation Assay, shRNA

Journal: International Journal of Molecular Medicine

Article Title: CENPU promotes tumorigenesis and stem cell properties in triple-negative breast cancer by suppressing lysosomal furin degradation

doi: 10.3892/ijmm.2025.5649

Figure Lengend Snippet: CENPU enhances the activity of furin. (A) Western blotting was performed to determine the expression levels of furin in MDA-MB-231, Cal51 and T47D cells. (B) Furin activity in MDA-MB-231, Cal51 and T47D cells was measured based on fluorescence intensity. (C) Michaelis constant values in the experimental group and control group of MDA-MB-231, Cal51 and T47D cells were obtained using double reciprocal plotting. (D) Mammosphere formation assays were performed after treatment with a furin inhibitor, and the number of tumor spheres per 200 cells was quantified (scale bar, 100 μ m). ** P<0.01. CENPU, centromere protein U; sh, short hairpin RNA; NS, not significant; RFU, relative fluorescence units; V, reaction velocity; S, substrate concentration.

Article Snippet: Human MDA-MB-231 (TNBC) and T47D (BC) cells were acquired from the American Type Culture Collection.

Techniques: Activity Assay, Western Blot, Expressing, Fluorescence, Control, shRNA, Concentration Assay