Journal: Nature Communications

Article Title: RNA-guided RNA silencing by an Asgard archaeal Argonaute

doi: 10.1038/s41467-024-49452-1

Figure Lengend Snippet: a HrAgo1 associates with 5’ phosphorylated (5’ P) small RNAs in vivo from E. coli . Nucleic acids that co-purified with HrAgo1 were [γ- 32 P] labeled, treated with RNase A or DNase I, and resolved on a denaturing gel (15% polyacrylamide 7 M urea). nt: nucleotides. b Length distribution of small RNAs associated with HrAgo1 as determined by small RNA sequencing. c Small RNAs associated with HrAgo1 have a bias for uracil bases at the 5’ end. d Sequences of guide and target oligonucleotides used in in vitro cleavage assays. e HrAgo1 cleaves ssRNA (but not ssDNA) targets with ssRNA guides, and ssDNA guides at lower efficiency, in the presence of Mg 2+ . HrAgo1 was incubated with ssDNA or ssRNA guides and Cy5-labeled ssDNA or RNA targets. Cy5-labeled cleavage products were resolved through denaturing (7 M urea) polyacrylamide gel electrophoresis and visualized by fluorescence imaging. Both ssRNA and ssDNA targets are 45nt. The HrAgo1-bound RNA extraction and digestion was carried out once; the results of the cleavage assays were confirmed by at least three repetitions.

Article Snippet: A plasmid suitable for expression of a HrAgo1 catalytic double-mutant (D585A & E623A; HrAgo1 DM ) was generated by Quikchange Site-Directed Mutagenesis using primers oPB199 and oPB201 for D585A and oPB200 and oPB198 for E623A, using E. coli strain NEB 5-alpha (New England Biolabs) (Table ). pX-sfGFP vector was a kind gift from Prof. Jae-Sung Woo (Korea University, South Korea).

Techniques: In Vivo, Purification, Labeling, RNA Sequencing Assay, In Vitro, Incubation, Polyacrylamide Gel Electrophoresis, Fluorescence, Imaging, RNA Extraction

Journal: Scientific Reports

Article Title: Versatile cell-based assay for measuring DNA alkylation damage and its repair

doi: 10.1038/s41598-021-97523-w

Figure Lengend Snippet: Schematic outline of alk-BER assay. The assay involves exposing the cells to MMS (step 1), isolation of total genomic DNA (step 2), conversion of MMS-induced methylated bases to SSBs with damage specific enzymes AAG and APE1 (step 3), separation of DNA fragments containing SSBs by alkaline agarose gel electrophoresis (step 4), gel staining, imaging, and quantitation of MDAs (step 5).

Article Snippet: Next, fresh pre-warmed media were added and cells were allowed to repair for 0, 3, 8, or 22 h. GM 12878 cells were treated with 5 mM (0.05%) MMS for 5 min. Total genomic DNA was purified from each time point using the PureLink genomic DNA mini kit (K 182001, Thermo Fisher Scientific).

Techniques: Isolation, Methylation, Agarose Gel Electrophoresis, Staining, Imaging, Quantitation Assay

Journal: Scientific Reports

Article Title: Versatile cell-based assay for measuring DNA alkylation damage and its repair

doi: 10.1038/s41598-021-97523-w

Figure Lengend Snippet: Alk-BER assay in yeast cells ( S. cerevisiae ). ( A ) Representative alkaline agarose gel image of MMS-induced DNA damage dose response in the BY4741 strain of S. cerevisiae . Genomic DNA of cells not exposed to MMS (C: control), and DNA of cells exposed to increasing doses of MMS (5, 10, or 20 mM) was resolved on alkaline agarose gel. Each DNA sample was treated with (+) and without (−) a cocktail of AAG and APE1 enzymes. ( B ) Dose dependent increase in the numbers of MMS-induced methyl G, A per 1 kb DNA fragment. Each data point denotes the average value and standard deviation of three independent experiments. ( C ) Representative gel image of DNA damage and repair time course in the BY4741 strain of S. cerevisiae . M: DNA size standard lambda/HindIII. C: control, cells not exposed to MMS, 0: cells collected after 10 min exposure to 20 mM MMS, 1–3 h: cells collected after 1, 2, 3 h of repair. ( D ) Quantitative representation of data displayed in panel C. Formation and repair of MMS-induced methyl G and A (7meG, 3meA), as a function of repair time. Each data point represents an average of 3 independent experiments; error bars were calculated based on standard deviation. Gel image presented in panel ( A ) has been cropped. Original, uncropped gel image is included in the supplementary data.

Article Snippet: Next, fresh pre-warmed media were added and cells were allowed to repair for 0, 3, 8, or 22 h. GM 12878 cells were treated with 5 mM (0.05%) MMS for 5 min. Total genomic DNA was purified from each time point using the PureLink genomic DNA mini kit (K 182001, Thermo Fisher Scientific).

Techniques: Agarose Gel Electrophoresis, Standard Deviation

Journal: Scientific Reports

Article Title: Versatile cell-based assay for measuring DNA alkylation damage and its repair

doi: 10.1038/s41598-021-97523-w

Figure Lengend Snippet: Alk-BER assay in human cells. ( A ) MMS dose response in SW13 cells. Cells were treated with increasing doses of MMS for 10 min at RT. Representative alkaline agarose gel image is shown. ( B ) Quantification of methyl A, G per 1 kb DNA fragment as a function of increasing MMS dose. The graph represents quantification of the data in panel ( A ). ( C ) Efficiency of double enzyme (AAG&APE1), and single enzymes: (AAG only), and (APE1 only), in converting methyl DNA adducts to SSBs. ( D ) Alkaline gel image representing DNA damage and repair time course. SW13 cells were exposed to 10 mM MMS for 10 min. Genomic DNA was isolated, and processed with double enzyme AAG&APE1 digest. ( E ) Quantification of the repair and removal of methyl A,G as a function of time. ( F ) SW13 cell viability measured by trypan blue. Each data point represents an average of 3 independent experiments; error bars were calculated based on standard deviation. Gel image presented in panel ( C ) has been cropped. Original, uncropped gel image is included in the supplementary data.

Article Snippet: Next, fresh pre-warmed media were added and cells were allowed to repair for 0, 3, 8, or 22 h. GM 12878 cells were treated with 5 mM (0.05%) MMS for 5 min. Total genomic DNA was purified from each time point using the PureLink genomic DNA mini kit (K 182001, Thermo Fisher Scientific).

Techniques: Agarose Gel Electrophoresis, Isolation, Standard Deviation

Journal: Scientific Reports

Article Title: Versatile cell-based assay for measuring DNA alkylation damage and its repair

doi: 10.1038/s41598-021-97523-w

Figure Lengend Snippet: The repair of MDAs is slow in human cells and does not correlate well with the levels of endogenous AAG enzyme. DNA damage and repair time course experiment was performed in several human cell lines; CHON-002, SW13, and HAP1. Cells (60–70% confluent) were exposed to 10 mM (0.1%) MMS in 1xPBS for 10 min at RT, followed by DNA repair time course 0, 3, 8 and 22 h at 37 °C. ( A ) DNA repair rates were quantitated individually for each cell line and expressed as a percentage (%) of the removed methyl A,G, as compared to the 0 h time point. Each data point represents an average of two independent experiments. ( B ) Endogenous levels of AAG enzyme were detected by Western blotting. Western blot image presented in panel ( B ) has been cropped. Original, uncropped blot image is included in the supplementary data.

Article Snippet: Next, fresh pre-warmed media were added and cells were allowed to repair for 0, 3, 8, or 22 h. GM 12878 cells were treated with 5 mM (0.05%) MMS for 5 min. Total genomic DNA was purified from each time point using the PureLink genomic DNA mini kit (K 182001, Thermo Fisher Scientific).

Techniques: Western Blot

Journal: Canadian Respiratory Journal

Article Title: Role of Erythromycin-Regulated Histone Deacetylase-2 in Benign Tracheal Stenosis

doi: 10.1155/2020/4213807

Figure Lengend Snippet: The expression of HDAC2 in the different groups determined by immunofluorescence. Image scale bar, 100 µ m. Red fluorescence (Cy3 staining) for the detection of the target protein HDAC2, blue fluorescence (DAPI staining) for the nucleus. Data are presented as the mean ± standard deviation in the corresponding histogram. ∗ P < 0.05 vs. the control group. Control: rabbit tracheal stenosis model without treatment; NS: rabbit tracheal stenosis model treated with penicillin; ERY: rabbit tracheal stenosis model treated with erythromycin; Budesonide: rabbit tracheal stenosis model treated with budesonide; Vorinostat: rabbit tracheal stenosis model treated with vorinostat. HDAC2, histone deacetylase-2; IF, immunofluorescence.

Article Snippet: Erythromycin enteric-coated tablets (H42021990, Yichang Humanwell Pharmaceutical Co., LTD.); Vorinostat Capsules (180509, Beijing Hengrui Kangda Medical Science and Technology Development Co., Ltd.); Budesonide (AstraZeneca 8339000); Rabbit Anti-Collagen III Polyclonal Antibody (bs-10423R, Bioss); Rabbit Anti-Collagen I Polyclonal Antibody (bs-0549R, Bioss); Rabbit Anti-HDAC2 Polyclonal Antibody (bs-1813R, Bioss); Rabbit VEGF ELISA kit (MM-021001); Rabbit TGF- β 1 ELISA kit (MM-3684001); Rabbit Polyclonal Anti-VEGF (bs-1313R, Bioss, 1/500–1/2000); Rabbit Polyclonal Anti-TGF β 1 (bs-0086R, Bioss, 1/500–1/2000); Rabbit monoclonal Anti-IL-8 (ab34100, abcam, 1/1000); Rabbit Polyclonal Anti-HDAC2 (OmnimAbs, OM105905, 1/500–1/2000); fluorescence microscope (CKX53, OLYMPUS); Microplate Reader (RT-6100, Rayto); Protein vertical electrophoresis instrument (DYY-6C, Beijing 61 instrument factory); Ultra High Sensitivity Chemiluminescence Imaging System (Chemi DocTM XRS+, Bio-Rad Shanhhai Laboratories).

Techniques: Expressing, Immunofluorescence, Fluorescence, Staining, Standard Deviation, Histone Deacetylase 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: Tissue Engineering. Part A

Article Title: Opposite Spectrum of Activity of Canonical Wnt Signaling in the Osteogenic Context of Undifferentiated and Differentiated Mesenchymal Cells: Implications for Tissue Engineering

doi: 10.1089/ten.tea.2010.0133

Figure Lengend Snippet: (A) QRT-PCR showing the expression profiles of three downstream targets of canonical Wnt signaling, axin2, myc, and cyclin D1. Quantified mRNA values were normalized by the amounts of glyceraldehyde 3-phophate dehydrogenase mRNA, and results are given as fold induction (*p < 0.05). (B) Immunoblotting analysis of β-catenin performed on nuclear fractions revealed higher levels of nuclear β-catenin in ASCs, E16, and FpN1 cells. Membranes were stripped and reprobed with anti-Lamin B1 antibody to assess for equal loading and transfer of nuclear proteins fraction. Histogram represents the densitometric analysis of electrophoresis bands, and the relative intensities of bands were normalized to their respective loading control and set as 100%. The results are presented as the mean ± standard deviation of three independent experiments. (C) Indirect immunofluorescence staining detected the most intense nuclear staining in ASCs, E16, and FpN1. As negative control normal primary (irrelevant) mouse immunoglobulin G was used. Nuclear counterstaining was performed with DAPI. QRT-PCR, quantitative reverse transcription–polymerase chain reaction; mASCs, mouse adipose-derived stem cells; E16, embryonic-stage day 16 calvarial mesenchymal cells; FpN1, postnatal day 1 frontal bones-derived osteoblast; PpN1, postnatal day 1 parietal bone-derived osteoblast; FpN60, postnatal day 60 frontal bone-derived osteoblast; PpN60, postnatal day 60 parietal bone-derived osteoblast; DAPI, 4′,6-diamidino-2-phenylindole; Wnt, wingless. Color images available online at www.liebertonline.com/ten.

Article Snippet: Nuclear counterstaining was performed using Vectashield H-1200 mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories), and a Zeiss Axioplan microscope equipped with an Axiocam HRc digital camera was used for imaging.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Electrophoresis, Standard Deviation, Immunofluorescence, Staining, Negative Control, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: The FEBS journal

Article Title: A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprin.

doi: 10.1111/j.1742-4658.2008.06592.x

Figure Lengend Snippet: Fig. 1. Simple 2D IEF ⁄ SDS ⁄ PAGE-based image analysis procedure. The procedure is based on qualitative differences among reference gels (level 1 match-sets) of each group of five gel replicates (three pooled biological gel replicates and two more technical gel repli- cates). Gel replicates of each group (activated meprin versus non-activated meprin) were cut virtually into four equally spaced quadrants for four independent image analyses. Reference gels of each group were then clustered into a new set for higher-level image analysis. The spot matching features of PDQUEST (version 7.3.1) allowed for detection of unique protein spots. The combined higher- level match-set is the final fusion of all annotated unique spots into one big 2D reference map.

Article Snippet: 2D IEF ⁄ SDS ⁄PAGE-based image analysis was performed using the program pdquest, version 7.3.1 (Bio-Rad Laboratories).

Techniques:

Journal: The FEBS journal

Article Title: A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprin.

doi: 10.1111/j.1742-4658.2008.06592.x

Figure Lengend Snippet: Fig. 2. Application of a simple 2D IEF ⁄ SDS ⁄ PAGE-based protease proteomic approach in substrate finding. A representative image analysis of the first quadrant is shown. Two hundred and fifty micrograms of conditioned medium protein from trypsin activated and non-activated MDCKa ⁄ b cells was separated by IEF in a 24 cm long IPG pH 3–10 NL strip. Vertical separation was according to mass in a 12.5% SDS gel. Optimized Ruthenium staining: for each condition (activated meprin versus non-activated meprin), three pooled biological gel replicates (from 18 dishes per pooled sample) and two more technical gel replicates (of one pooled sample) were produced for subsequent image analysis. Unique protein spots are labelled in level 1 and higher-level match-sets with SSP assigned by the image analysis software.

Article Snippet: 2D IEF ⁄ SDS ⁄PAGE-based image analysis was performed using the program pdquest, version 7.3.1 (Bio-Rad Laboratories).

Techniques: Stripping Membranes, SDS-Gel, Staining, Produced, Software