Journal: Nature

Article Title: Asymmetric Cell Divisions Promote Notch-Dependent Epidermal Differentiation

doi: 10.1038/nature09793

Figure Lengend Snippet: a, Immunodetection of anaphase-telophase pronuclei. with spindle midbody marker Survivin. b, Apical colocalisation of ACD components during mitosis. c,d shRNA knockdown efficiencies in keratinocytes and epidermis (n=3 separate experiments). e, Representative axes of division (lines) in E16.5 transduced anaphase/telophase cells. f, Radial histogram quantification of data from (e), n’s are indicated. g, Cell-autonomous elimination of ACDs upon LGN , Numa1 , or Dctn1 knockdown. h–i, Interdependence of Gαi3/LGN/NuMA cortical localisation. j,k Misalignment of angles between LGN crescent centre and centrosomal axis (spindle) upon Numa1 knockdown (each dot indicates a single data point). Scale bars: 10µm. Error bars: S.D. ( c, d ); S.E.M ( k ). Dotted lines denote basement membrane (thick); cell boundaries (thin).

Article Snippet: Like transgenic mInsc , lentiviral EYFP-mInsc increased ACDs (p=0.0196 by Chi-square).

Techniques: Immunodetection, Marker, shRNA, Knockdown, Membrane

Journal: Nature

Article Title: Asymmetric Cell Divisions Promote Notch-Dependent Epidermal Differentiation

doi: 10.1038/nature09793

Figure Lengend Snippet: a,b, Reduced terminal differentiation in E17.5 ACD knockdowns. Basally-transduced regions are identified by H2B-mRFP1, always most intense in suprabasal progeny. Note correlation of repressed differentiation with transduction (RFP + ; line demarcates low/high infection boundary). c,d, Partial restoration of shLGN-1617 epidermal defects upon transducing full-length(FL) LGN or LGNΔC (n>15 fields; n>6 embryos/condition). e–h, EYFP-mInsc enhancement of LGN-dependent ACDs. e, EYFP-mInsc and LGN immunolocalisation in mitotic cells of E17.5 shScramble or shLGN-1617 epidermis after EYFP-mInsc . co-transduction. f, Quantifications of division axes (n’s indicated). ( g,h ) LGN-dependent enhancement of spinous-layer thickness upon mInsc overexpression (n>10 fields; n>3 embryos/condition). Scale bars: 50 µm ( a–c , g ), 10 µm ( e ). Error bars are S.E.M.

Article Snippet: Like transgenic mInsc , lentiviral EYFP-mInsc increased ACDs (p=0.0196 by Chi-square).

Techniques: Transduction, Infection, Over Expression

Journal: Nature

Article Title: Asymmetric Cell Divisions Promote Notch-Dependent Epidermal Differentiation

doi: 10.1038/nature09793

Figure Lengend Snippet: a, qPCR vs. microarray comparisons of Notch pathway gene expression in E14–E15 wild-type epidermis. b,c, Diminished Hes1 and full-length Notch3 in shLGN-1617 -transduced epidermis. Line (b) demarcates low/high-infection boundary. d, Decreased Notch3 (p=0.0133) and Hes1 (p=0.0169) mRNAs in E18 shLGN-1617 suprabasal cells. Note also dampened suprabasal:basal Notch1 (p=0.20), Notch2 (p=0.19). e, Lentiviral Notch reporter for coordinate shRNA-knockdown. f,g Abrogation of Notch reporter expression (EGFP + ), concomitant with differentiation defects, in E17.5 RBPJ cKO and shLGN-1617 epidermis. h, Effects of LGN/Numa1 knockdown on Notch reporter activity (n>24 fields; >3 embryos/condition). i , Reduced activity in P0 Notch reporter transgenics transduced with shLGN-1617;H2B-mRFP1 . Error bars in a,d represent S.D; S.E.M. in h . Scale bars: 50µm. For qPCR ( a,d ), n’s are triplicates from 2 separate experiments.

Article Snippet: Like transgenic mInsc , lentiviral EYFP-mInsc increased ACDs (p=0.0196 by Chi-square).

Techniques: Microarray, Gene Expression, Infection, shRNA, Knockdown, Expressing, Activity Assay, Transduction

Journal: Nature

Article Title: Asymmetric Cell Divisions Promote Notch-Dependent Epidermal Differentiation

doi: 10.1038/nature09793

Figure Lengend Snippet: a–c, Normal LGN localization and ACDs in RBPJ mutants (each dot represents one data point in b , n’s indicated in c ). d , e, Analyses of differentiation defects in E17.5 headskins from control or RBPJ fl/fl embryos transduced at E9.5 with shLGN-1617;H2B-mRFP1 (LGN1617), shScramble;NLS-Cre-mRFP1 ( RBPJ +scramble), or shLGN-1617;NLS-Cre-mRFP1 ( RBPJ +LGN1617). Comparable defects in double and single mutants/knockdowns, suggest a common pathway for RBPJ and LGN . f–h, Restoring Notch signalling rescues shLGN-1617 differentiation defects. Headskin ( f ); backskin ( g,h ). Combinations of single and double mutant clones (separated by vertical lines) expressing shLGN-1617 (red) and active NICD (GFP, pseudocolored in blue) were generated by co-infecting E9.5 Rosa-Lox-stop-Lox-NICD-IRES-GFP -knockin embryos with shScramble/shLGN-1617;H2B-mRFP1 and NLS-Cre . Scale bars: 10µm ( a ); 50µm ( d, f, g ). Error bars represent S.D. ( b ), S.E.M. ( e,h ). p values from two-tailed student’s t-tests are indicated; ns: not statistically significant. For e , h , n>10 fields; n>3 embryos.

Article Snippet: Like transgenic mInsc , lentiviral EYFP-mInsc increased ACDs (p=0.0196 by Chi-square).

Techniques: Control, Mutagenesis, Clone Assay, Expressing, Generated, Knock-In, Two Tailed Test

Journal: bioRxiv

Article Title: Reverse engineering of a pathogenic antibody reveals the molecular mechanism of vaccine-induced immune thrombotic thrombocytopenia

doi: 10.1101/2023.06.30.547300

Figure Lengend Snippet: Surface-plasmon resonance sensograms obtained for the binding of the PF4 to the RVT1-captured Protein A chip. The concentration of the PF4 was titrated from 100nM till 2uM concentrations, and the signal was normalized to the sensograms with the blank solution whose composition matches the solution of PF4.

Article Snippet: The Protein A conjugated chip was acquired from Xantec Ltd (Düsseldorf, Germany).

Techniques: SPR Assay, Binding Assay, Concentration Assay

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: PCV2 infection led to translocation of HMGB1 from nuclei to cytoplasmic compartments. PK-15 cells and porcine monocytic cells (3D4/31) were infected for 36 h with PCV2 (MOI = 1) or mock infected as a control. (A) Confocal imaging of HMGB1 distribution in PCV2-infected cells immunostained with anti-HMGB1 (green) and anti-Cap (red) antibodies. Nuclei were labeled with DAPI (blue). Representative micrographic images are shown. (B) Immunoblotting of PCV2 Cap and HMGB1 in nuclear and cytoplasmic extracts from PCV2- or mock-infected PK-15 cells. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic fractions, respectively. (C) The intensity of protein bands was quantified densitometrically using Gel-Pro Analyzer. Ratios of nuclear or cytoplasmic HMGB1 to Histone H3 or GAPDH were quantified, respectively. (D and E) Quantification of hmgb1 mRNA by qPCR in PK-15 and 3D4/31 cells infected with PCV2 for different times using total RNA extracts from the cells. (F and G) Immunoblotting of HMGB1 and PCV2 Cap in the lysates of PK-15 and 3D4/31 cells infected with PCV2 for different times. β-Actin was used as a loading control. The data in panels A, B, F, and G are representative of three independent experiments. Bar charts in panels C, D, and E show means ± SDs from three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Translocation Assay, Imaging, Labeling, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Overexpression of HMGB1 inhibited PCV2 replication. PK-15 cells were transfected with recombinant plasmid expressing HMGB1 (pHMGB1) or control plasmid (pFlag) for 24 h and then infected with PCV2 (MOI = 1) for 36 h. (A) Effect of HMGB1 overexpression on PCV2 Cap expression as shown by immunoblotting using protein samples from the whole-cell lysates. β-Actin was used as a loading control. The gel shown is representative of three independent experiments. (B) The ratios of band intensity of HMGB1 or PCV2 Cap to β-actin (as shown in panel A). (C) Effect of HMGB1 overexpression on PCV2 orf2 (encoding Cap) transcription measured by qPCR using total RNA extracted from the whole-cell lysates. (D) PCV2 replication in cells overexpressing HMGB1 as assessed by indirect immunofluorescence. Percentages of PCV2-infected cells were calculated as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the HMGB1 overexpressing cells are shown with nontransfected but PCV2-infected cells set at 100%. (E) PCV2 genomic DNA copies in cells overexpressing HMGB1 quantified by qPCR using total DNA extracts from whole-cell lysates. Bar charts in panels B, C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01; ***, P < 0.001.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Over Expression, Transfection, Recombinant, Plasmid Preparation, Expressing, Infection, Western Blot, Immunofluorescence

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Downregulation of HMGB1 promoted PCV2 replication. PK-15 cells were transfected with hmgb1-specific RNA interference (RNAi) plasmid (sh-HMGB1) or control RNAi plasmid (sh-NC) for 24 h and then infected with PCV2 (MOI= 1) for 36 h. (A) Effect of hmgb1 knockdown on PCV2 Cap expression (β-actin used as a loading control) as shown by immunoblotting using protein samples from the whole-cell lysates. The gel shown is representative of three independent experiments. (B) The ratios of band intensity of HMGB1 or PCV2 Cap to β-actin (as shown in panel A). (C) Effect of hmgb1 knockdown on PCV2 orf2 (encoding Cap) transcription examined by qPCR using total RNA extracted from the whole-cell lysates. (D) PCV2 replication in hmgb1-silenced cells as assessed by indirect immunofluorescence. Representative fluorescence images are shown (top). Percentage of PCV2-infected cells was calculated by dividing the number of PCV2-infected cells by the total cell number in each group (n = 2 images for each experiment per group) that were counted using ImageJ software. Relative percentages of PCV2-infected cells in the hmgb1-silenced cells are shown with nontransfected but PCV2-infected cells set at 100% (bottom). (E) Effect of hmgb1 silencing on PCV2 genomic DNA copies measured by qPCR using total DNA extracts from whole-cell lysates. Bar charts in panels B, C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Transfection, Plasmid Preparation, Infection, Expressing, Western Blot, Immunofluorescence, Fluorescence, Software

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Nuclear HMGB1 repressed PCV2 replication. PK-15 cells were transfected with recombinant plasmid expressing HMGB1 (pHMGB1) or control plasmid (pFlag) for 24 h and then infected with PCV2 (MOI = 1) for 36 h. Nuclear and cytoplasmic extracts were prepared for immunoblotting as described in the legend for Fig. 1 Immunoblotting of HMGB1 and PCV2 Cap in the nuclear (A) and cytoplasmic (B) fractions. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic extracts, respectively. Representative images from three independent experiments are shown. The ratios of band intensities of HMGB1 or PCV2 Cap to those of histone H3 (as shown in panel A) in the nuclear fraction (C) or to GAPDH (as shown in panel B) in the cytoplasmic fraction (D). (E) Effect of HMGB1 overexpression on PCV2 orf2 transcription in the nuclei examined by qPCR using total RNA extracted from the nuclear fractions. Results were normalized to histone H3 mRNA in the same samples. (F) PCV2 genomic DNA replication in the nuclei of HMGB1-overexpressing cells quantified by qPCR using total DNA extracted from nuclear fractions. Bar charts in panels C to F show means ± SDs from three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Transfection, Recombinant, Plasmid Preparation, Expressing, Infection, Western Blot, Over Expression

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Ethyl pyruvate inhibited nucleocytoplasmic translocation of HMGB1 in PCV2-infected cells. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) with or without ethyl pyruvate (EP; 7.5 mM) treatment. The cell samples were harvested at 36 hpi. (A) Confocal microscopic images show inhibition of nuclear HMGB1 migration into the cytosol by EP. Cells were immunostained for HMGB1 (green) and PCV2 Cap (red), with nuclei stained with DAPI (blue). Bars, 10 μm. (B) Immunoblotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic fractions of PCV2-infected and EP-treated cells. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic extracts, respectively. Representative images from three independent experiments are shown. The ratios of band intensities of HMGB1 or PCV2 Cap to those of histone H3 (as shown in panel B, left) in the nuclear fraction (C) or to GAPDH (as shown in panel B, right) in the cytoplasmic fraction (D). (E) Effect of EP on PCV2 genomic DNA replication by qPCR using DNA extracted from nuclei of PCV2-infected cells treated with 7.5 mM EP. Bar charts in panels C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Infection, Inhibition, Migration, Staining, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: N-Acetylcysteine inhibited PCV2-induced HMGB1 translocation from nuclei to cytosol and repressed PCV2 replication. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) for 12 h and then treated with 10 mM N-acetylcysteine (NAC). The cell samples were harvested at 36 hpi. (A) Confocal imaging of HMGB1 distribution in PCV2-infected and NAC-treated cells after the cells were fixed and immunostained for HMGB1 (green) and Cap (red). Nuclei were stained with DAPI (blue). Bars, 10 μm. (B) Blotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic extracts of PCV2-infected cells with or without NAC treatment. Histone H3 and GAPDH were used as internal controls for the nuclear and cytoplasmic fractions, respectively. The figure is representative of three independent experiments. The ratios of band intensities of HMGB1 or PCV2 Cap to histone H3 (as shown in panel B, left) in the nuclear fraction (C) or to GAPDH (as shown in panel B, right) in the cytoplasmic fraction (D). (E) Effect of NAC on PCV2 genomic DNA replication by qPCR using DNA extracted from lysates of PCV2-infected cells treated with NAC. (F) Cytosolic ROS levels in PCV2-infected cells with or without treatment by NAC or ethyl pyruvate (EP) as measured by flow cytometry after probing with DCFH-DA. Bar charts in panels C, D, E, and F show means ± SDs from three independent experiments. *, P < 0.05; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Infection, Imaging, Staining, Flow Cytometry

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Ethyl pyruvate was inhibitory to PCV2 infection. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) with or without ethyl pyruvate (EP; 7.5 mM) treatment. The cell samples were harvested at 36 hpi. (A) Effect of EP on PCV2 replication in PK-15 cells by immunofluorescence. Percentages of PCV2-infected cells were calculated as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the EP-treated cells are shown with untreated but PCV2-infected cells set at 100%. (B) Immunoblotting of HMGB1 and PCV2 Cap in whole-cell lysates with β-actin used as a loading control. (C) The ratios of band intensities of HMGB1 or PCV2 Cap to β-actin (as shown in panel B). Bar charts in panels A and C show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Immunofluorescence, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: The B box domain of HMGB1 was involved in inhibition of PCV2 replication. (A) Schematic illustration of the full-length and truncated forms of porcine HMGB1 according to its human homolog. All truncated versions, A box, AB box, and B box plus C terminus (B boxCT), were flag tagged. The numbers indicate positions of amino acids. Arrows with C followed by numbers represent key cysteine residues. NLS, nuclear localization signal. PK-15 cells were transfected with recombinant plasmids expressing flag-tagged or full-length HMGB1 for 24 h and then infected with PCV2 (MOI = 1) for 36 h. (B) Numbers of PCV2-infected cells examined by immunofluorescence using anti-Cap monoclonal antibody as the probe (top). Expression of PCV2 Cap and different forms of HMGB1 as assessed by immunoblotting using the whole-cell lysates harvested at 36 hpi and antibodies against Flag, Cap, and HMGB1 (bottom). β-Actin was used as a loading control. The panel B images are representative of three individual experiments. (C) The ratios of band intensities of PCV2 Cap to those of β-actin (as shown at the bottom panel of B). (D) Effect of different HMGB1 truncations on PCV2 DNA replication estimated by qPCR using total DNA extracted from the whole-cell lysate. Bar charts in panels C and D show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Inhibition, Transfection, Recombinant, Expressing, Infection, Immunofluorescence, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: HMGB1 bound to the Ori region of the PCV2 genome. (A) Binding of porcine HMGB1 to PCV2 DNA using the gel shift assay. PCV2 DNA (500 ng) and various concentrations (0 to 5 μg) of purified His-tagged recombinant HMGB1 were mixed in binding buffer. The DNA-protein mixtures were subjected to 0.8% agarose gel electrophoresis to visualize changes of the DNA motility. (B) Binding of HMGB1 to a specific region of PCV2 DNA: full-length and different fragments of PCV2 genome (orf1, orf2, and Ori) were incubated with recombinant HMGB1 protein to identify the region of PCV2 genome involved in HMGB1 binding. (C) To confirm the Ori region is required for HMGB1 binding, the Ori fragment was combined with orf1 or orf2 (Ori-orf1 or Ori-orf2) that were then compared with orf1 or orf2 alone by the gel shift assay. (D) Immunoprecipitation of purified HMGB1 protein (500 μg) and PCV2 DNA (500 ng) mixture by anti-HMGB1 antibody (rabbit IgG as control) and protein A/G agarose. The precipitates were probed with anti-His and anti-HMGB1 antibodies by immunoblotting. (E) Quantification of PCV2 genomic Ori copies by qPCR in DNA extracts from PCV2 DNA-HMGB1 precipitates (shown in panel D) after DNase pretreatment. (F) Blotting of HMGB1 in immunoprecipitates of whole-cell lysates (WCL) of the PK-15 cells infected with PCV2 (36 h) by anti-HMGB1 (rabbit IgG as control) and protein A/G agarose. (G) Quantification of PCV2 genomic Ori copies by qPCR in DNA extracts from immunoprecipitates of whole-cell lysates (shown in panel F) after DNase pretreatment. (H) Blotting of HMGB1 in immunoprecipitates of nuclear extracts of the PK-15 cells infected with PCV2. (I) Quantification of PCV2 genomic Ori copies in the precipitates shown in panel H. Bar charts in panels E, G, and I show means ± SDs from three independent experiments.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Purification, Recombinant, Agarose Gel Electrophoresis, Incubation, Immunoprecipitation, Western Blot, Infection

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Effect of hydrogen peroxide treatment on subcellular localization of HMGB1 and PCV2 replication. (A) H2O2 treatment promoted nucleocytoplasmic translocation of HMGB1. PK-15 cells were treated with or without N-acetylcysteine (NAC; 10 mM) before adding 50 μM H2O2. Cells were fixed and immunostained with anti-HMGB1 (green) for confocal microscopy. Nuclei were labeled with DAPI (blue). (B) Immunoblotting of HMGB1 in the nuclear and cytoplasmic fractions of PK-15 cells treated with H2O2 and NAC. (C) Confocal imaging of PK-15 cells infected by PCV2 with or without 50 μM H2O2 treatment after immunostaining with anti-HMGB1 (green) and anti-Cap (red) antibodies. (D) Blotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic extracts of PCV2-infected cells with or without H2O2 treatment. Histone H3 and GAPDH were used as internal controls for the nuclear and cytoplasmic fractions, respectively. (E) Percentages of PCV2-infected cells were calculated from immunofluorescence images as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the H2O2-treated cells are shown with nontreated but PCV2-infected cells set at 100%. Bar chart in panel E shows means ± SDs from three independent experiments. **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Confocal Microscopy, Labeling, Western Blot, Imaging, Infection, Immunostaining, Immunofluorescence

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Schematic illustration of the interaction between PCV2 and HMGB1 in infected cells. HMGB1 in the nucleus restricts PCV2 replication by binding to the Ori region of the PCV2 genome. PCV2 infection causes increased generation of cellular ROS. Increased ROS promotes nucleocytoplasmic translocation of HMGB1 and lessens sequestration of the viral DNA by HMGB1 in the nucleus, thus enhancing PCV2 replication. N-Acetylcysteine (and probably ethyl pyruvate as well) scavenges PCV2-induced ROS and thus increases retention of HMGB1 in the nucleus, leading to sequestration of viral DNA and reduced PCV2 replication.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Binding Assay, Translocation Assay

Journal: The Journal of Biological Chemistry

Article Title: The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

doi: 10.1016/j.jbc.2022.101699

Figure Lengend Snippet: rs4903064 resides in a renal cancer–associated regulatory element. A , ATAC-seq tracks from primary renal cancer cells at the DPF3 locus. The top track indicates SNPs in high LD with rs4903064 (r 2 > 0.8). ATAC-seq was performed in cells from three individuals with different rs4903064 genotypes (TT, CT, CC). KIRC RE indicates r egulatory e lements defined by ATAC-seq in ccRCC TCGA samples. B , zoomed in view of ( A ). SNP rs4903064 resides in open chromatin and in KIRC_67261. Please note that open chromatin is only detected in cells carrying the risk allele C. C , sequences of the two alleles at rs4903064. The risk allele C creates a putative hypoxia-responsive element (HRE). D , allele-specific qPCR assay for rs4903064 indicates a shift toward the risk allele C in DNA fragments captured by ATAC experiments in tumor cells ( red triangle ) or tubular cells exposed to 1 mM DMOG ( orange triangle ) when compared to input DNA or DNA from ATAC experiments in untreated tubular cells. Cells were isolated from an individual heterozygous for rs4903064. E , allelic ratio of rs4903064 in ATAC samples from tubular cells isolated from three individuals heterozygous for the SNP performed in triplicates. Cells were exposed to 1 mM DMOG for 16 h or left untreated. Bars indicate mean values ± SD. t test, ∗∗∗∗ p < 0.0001. F , allelic ratio of rs4903064 in FAIRE samples from cells of four heterozygous individuals. Cells were exposed to 1 mM DMOG for 16 h, and values were normalized to corresponding input DNA. The mean value for each individual is shown. Bars indicate mean values ± SD. ∗one sample t test, p < 0.05. G , reporter assay in HeLa cells using the pGL3 promoter vector with or without a 415 bp sequence covering the protective or the risk allele of rs4903064. Cells were treated with 1 mM DMOG for 16 h as indicated. Bars indicate mean values ± SD of one experiment performed in triplicates. H , HeLa cells were depleted for the indicated HIF-α isoforms using siRNA and transfected with the reporter plasmid including the risk allele. Cells were exposed to 1 mM DMOG for 16 h. Reporter activity was normalized to the activity of co-transfected β-galactosidase. Bars indicate mean values ± SD of one experiment performed in triplicates. I and J , DPF3 protein levels are high in HIF-positive ccRCC tumors. A total of 330 ccRCC tumors were stratified according to the immunoreactive score for HIF-1α, HIF-2α, and DPF3 staining in no, low, medium, or strong staining. Fractions of DPF3 signals are shown for each subgroup of HIF staining. Pearson χ 2 -test; p = 1.8 × 10 −8 for HIF-1α ( I ) and p = 0.003 for HIF-2α ( J ). ATAC, Assay for Transposase-Accessible Chromatin; ccRCC, clear cell renal cell carcinoma; DMOG, dimethyloxalylglycine; DPF3, double PHD fingers 3; FAIRE, formaldehyde-assisted isolation of regulatory elements; HIF, hypoxia-inducible transcription factor; KIRC, kidney renal clear cell carcinoma; SNP, single nucleotide polymorphism.

Article Snippet: ChIP experiments were performed as previously described using antibodies directed against HIF-1α (Cay10006421, rabbit polyclonal, Cayman Chemicals), HIF-1β (NB100–110, rabbit polyclonal, Novus Biologicals), RNApol 2 (SC-899, rabbit polyclonal, Santa Cruz), or H3K27ac (ab4729, rabbit polyclonal, Abcam) ( , ).

Techniques: Isolation, Reporter Assay, Plasmid Preparation, Sequencing, Transfection, Activity Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

doi: 10.1016/j.jbc.2022.101699

Figure Lengend Snippet: HIF binding to the RCC risk allele. A , HIF ChIP qPCR results for the DPF3 locus. Experiments were performed from cells isolated from 11 TT, 11 CT, and 1 CC individuals. Bars indicate mean values ± SD. Student’s t test, ∗ p < 0.05, ∗∗ p < 0.01. B , binding of HIF to the control locus at EGLN3 is not affected by the rs4903064 genotype. Bars indicate mean values ± SD. C , HIF ChIP-seq tracks in two different ccRCC cell lines at the rs4903064-associated enhancer (TT: RCC4 and CC: individual 40,911,432 from Yao et al. ). HIF binding is only detectable in cells from a patient carrying the C allele ( red , HIF-1α: 12 × C from 12 reads in total, HIF-2α 99 × C from 99 reads in total). D , same tracks as in ( C ) covering the EGLN3 control locus. E and F , H3K27ac ChIP-seq tracks from corresponding normal ( blue ) and ccRCC ( red ) tissue indicates increased activity of the disease-associated regulatory element at DPF3 ( E , highlighted in yellow ) and of the EGLN3 locus ( F ) in tumors. G , H3K27ac ChIP-seq tracks for two different ccRCC cell lines at the rs4903064-associated enhancer (TT: 786-O and CC: individual 40,911,432 from Yao et al. ). Levels of the active enhancer mark H3K27ac are high in the individual carrying the C allele and reduced upon VHL re-expression. H , same tracks as in ( G ) covering the EGLN3 control locus. Note that H3K27ac levels are reduced upon VHL re-expression in both cell lines. ccRCC, clear cell renal cell carcinoma; ChIP, chromatin immunoprecipitation; DPF3, double PHD fingers 3; EGLN3, Egl-9 family hypoxia inducible factor 3; HIF, hypoxia-inducible transcription factor; KIRC, kidney renal clear cell carcinoma; SNP, single nucleotide polymorphism; VHL, von Hippel-Lindau.

Article Snippet: ChIP experiments were performed as previously described using antibodies directed against HIF-1α (Cay10006421, rabbit polyclonal, Cayman Chemicals), HIF-1β (NB100–110, rabbit polyclonal, Novus Biologicals), RNApol 2 (SC-899, rabbit polyclonal, Santa Cruz), or H3K27ac (ab4729, rabbit polyclonal, Abcam) ( , ).

Techniques: Binding Assay, ChIP-qPCR, Isolation, Control, ChIP-sequencing, Activity Assay, Expressing, Chromatin Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

doi: 10.1016/j.jbc.2022.101699

Figure Lengend Snippet: Allele specific interactions at rs4903064. A – C , allele-specific qPCR on ChIP samples generated from primary tubular cells exposed to 1 mM DMOG for 16 h. A , HIF ChIP indicates enrichment for the C allele in DNA fragments captured with HIF-1α or HIF-1β antibodies. n = 7 individuals. B , H3k27ac as a marker for active chromatin is enriched at the C allele. n = 4 individuals. C , RNApol2 is enriched at the C allele of rs4903064 indicating increased interaction with the transcriptional machinery from this allele. n = 4 individuals. D , quantification of the results in ( A – C ). The allelic ratio is shifted to the risk allele C, which indicated increased interaction with HIF and the transcriptional machinery as well as increased activity of this allele. Values are mean ± SD. One sample t test, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ChIP, chromatin immunoprecipitation; DMOG, dimethyloxalylglycine; HIF, hypoxia-inducible transcription factor.

Article Snippet: ChIP experiments were performed as previously described using antibodies directed against HIF-1α (Cay10006421, rabbit polyclonal, Cayman Chemicals), HIF-1β (NB100–110, rabbit polyclonal, Novus Biologicals), RNApol 2 (SC-899, rabbit polyclonal, Santa Cruz), or H3K27ac (ab4729, rabbit polyclonal, Abcam) ( , ).

Techniques: Generated, Marker, Activity Assay, Chromatin Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

doi: 10.1016/j.jbc.2022.101699

Figure Lengend Snippet: Regulation of DPF3 expression and effect of DPF3 on tubular cell growth. A , relative expression levels of DPF3 in primary renal tubular cells exposed to 1 mM of DMOG for 16 h. Samples were stratified according to the rs4903064 genotype (TT = 42, CT/CC = 44). Values were normalized to expression levels of the housekeeping gene HPRT and to values from corresponding untreated cells. Each dot represents the mean value from cells of one individual. qPCR was performed in duplicates per individual. Bars indicate mean values ± SD. Student’s t test, ∗∗∗∗ p < 0.0001. B , DPF3 expression levels in isolated tumor cells compared to corresponding untreated tubular cells from 8 (TT) and 14 (CT/CC) individuals, respectively. Bars indicate mean values ± SD. One sample t test, ∗ p < 0.05. C , top , allelic ratios of rs4903064 in cells from heterozygous individuals exposed to DMOG 1 mM or left untreated. One sample t test, ∗∗∗ p < 0.001. Bottom , allelic ratios of rs4903064 in ccRCC cells with high (CA9+) or low (CA9−) CA9 expression as a marker for HIF-1α activity. Values are mean from technical triplicates, and each dot represents values derived from one individual. Bars indicate mean values ± SD. t test, ∗ p < 0.05. D , Western blot for DPF3 and beta actin from lysates of different clones of HKC-8 cells with or without defective DPF3. E , Western blot for DPF3 and beta actin from lysates of two different huPTC lines transfected either with nontargeting (nt) guides or guides targeting DPF3. F , proliferation assays of the clones of cells indicated in ( D ). Dark lines and dots indicate the mean value from the three different clones of cells, which were tested in biological triplicates. Light gray and light blue lines indicate individual values for the three different clones of cells. G , proliferation assay from cells indicated in ( E ). H , Western blot for DPF3 and beta actin from lysates of primary renal tubular cells (PTC) transfected either with nontargeting (nt) guides or guides targeting DPF3. Cells were harvested at day 3 after transfection. I , cyst volume of PTC 7 days after plating in a Matrigel matrix (n = 3 wells per condition, 4 quadrants per well). J , representative images of the cysts in the Matrigel matrix. Values are mean ± SD. t test, ∗ p < 0.05, ∗∗ p < 0.01 for F , G , and I . CA9, carbonic anhydrase 9; ccRCC, clear cell renal cell carcinoma; DMOG, dimethyloxalylglycine; DPF3, double PHD fingers 3; huPTC, human urinary primary tubular cells.

Article Snippet: ChIP experiments were performed as previously described using antibodies directed against HIF-1α (Cay10006421, rabbit polyclonal, Cayman Chemicals), HIF-1β (NB100–110, rabbit polyclonal, Novus Biologicals), RNApol 2 (SC-899, rabbit polyclonal, Santa Cruz), or H3K27ac (ab4729, rabbit polyclonal, Abcam) ( , ).

Techniques: Expressing, Isolation, Marker, Activity Assay, Derivative Assay, Western Blot, Clone Assay, Transfection, Proliferation Assay