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: Nature Communications

Article Title: Estrogen-related receptor γ causes osteoarthritis by upregulating extracellular matrix-degrading enzymes

doi: 10.1038/s41467-017-01868-8

Figure Lengend Snippet: Signaling upstream of ERRγ expression. a qRT-PCR analysis ( n ≥ 8) of IL-6 in chondrocytes treated with IL-1β and infected with 800 MOI of control virus (Ad-C) or the indicated MOI of Ad- Epas1 or Ad- Slc3918 . b , c Detection of IL-6 in chondrocytes treated with IL-1β (1 ng ml –1 ) by immunofluorescence microscopy ( b ) and Western blotting ( c ). d , e Western blot ( d ) and qRT-PCR ( e ) analysis of ERRγ, MMP3, and MMP13 in chondrocytes treated with IL-6 ( n = 10). MMP3 and MMP13 proteins in conditioned medium of chondrocyte cultures were detected by Western blotting. f qRT-PCR analysis ( n ≥ 8) of ERRγ mRNA levels in chondrocytes treated with IL-1β (left panel). qRT-PCR analysis ( n ≥ 8) of ERRγ mRNA levels in chondrocytes treated with IL-6 siRNA and exposed to IL-1β (middle panel). qRT-PCR analysis ( n ≥ 8) of ERRγ mRNA levels in chondrocytes treated with AG490 and exposed to IL-6 (right panel). Values are presented as means ± s.e.m. (* P < 0.05, ** P < 0.005, and *** P < 0.0005. One-way ANOVA)

Article Snippet: Additional immunostaining was performed using antibodies against ERRα (40 μg ml –1 ; sc-65715; Santa Cruz Biotech), MMP3 (4 μg ml –1 ; ab52915; Abcam), MMP13 (1:100 dilution; ab51072; Abcam), SOX9 (12 μg ml –1 ; ab185966; Abcam), and type II collagen (2 μg ml –1 ; MAB8887; Millipore).

Techniques: Expressing, Quantitative RT-PCR, Infection, Immunofluorescence, Microscopy, Western Blot

Journal: Nature Communications

Article Title: Estrogen-related receptor γ causes osteoarthritis by upregulating extracellular matrix-degrading enzymes

doi: 10.1038/s41467-017-01868-8

Figure Lengend Snippet: ERRγ directly upregulates MMP3 and MMP13 in chondrocytes. a The mRNA (left) and protein (right) levels of ERRγ, MMP3, and MMP13 in primary cultured chondrocytes infected with Ad-C (800 MOI) or the indicated MOI of Ad- Esrrg for 36 h ( n = 12). b Representative images showing immunostaining for MMP3, MMP13, SOX9, and type II collagen (Coll-II) in cartilage sections of mice obtained after 3 weekly IA injections with Ad-C or Ad- Esrrg ( n = 12 mice per group). c The ERRE sequences in the promoter regions of Mmp3 and Mmp13 are indicated. d Quantitative ChIP assays were performed, and the data are presented as fold-changes relative to each input ( n ≥ 4). e qRT-PCR ( n = 11) analyses of MMP3 and MMP13 expression in chondrocytes treated with IL-6 (100 ng ml –1 ) in the presence of the indicated concentrations of GSK5182, an inverse agonist of ERRγ. f Sham-operated or DMM-operated mice were IP-injected with GSK5182 and killed 8 weeks after the operation. Representative images of safranin-O staining and scoring of OARSI grade, osteophyte size, and subchondral bone plate thickness ( n = 10 mice per group). Values are presented as means ± s.e.m. (* P < 0.05, ** P < 0.005, and *** P < 0.0005). One-way ANOVA ( a , d , e ), two-tailed t test ( f ), and Mann–Whitney U test ( f ). Scale bar: 50 μm

Article Snippet: Additional immunostaining was performed using antibodies against ERRα (40 μg ml –1 ; sc-65715; Santa Cruz Biotech), MMP3 (4 μg ml –1 ; ab52915; Abcam), MMP13 (1:100 dilution; ab51072; Abcam), SOX9 (12 μg ml –1 ; ab185966; Abcam), and type II collagen (2 μg ml –1 ; MAB8887; Millipore).

Techniques: Cell Culture, Infection, Immunostaining, Quantitative RT-PCR, Expressing, Injection, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Cell reports

Article Title: Microglia are Indispensable for Synaptic Plasticity in the Spinal Dorsal Horn and Chronic Pain

doi: 10.1016/j.celrep.2019.05.087

Figure Lengend Snippet: (A) Spinal LTP at C-fiber synapses was induced by HFS at 10 and 20 V but not at 1V. The representative traces of C-fiber evoked fEPSPs were recorded before (i) and 3 h (ii) after high frequency stimulation (HFS) of the sciatic nerve at different intensities. Amplitude of C-fiber evoked fEPSPs (red vertical line) was determined automatically by parameter extraction software of WinLTP. Scale bars: x =100 ms, y = 0.2 mV. Summary data of the C-fiber responses expressed as mean ± SEM plotted vs. time were shown below (n = 6 mice/group). The arrow indicates the time point at which HFS was delivered. (B, C) Representative images and summary data show the expression of ATF3 in L4 DRG neurons 7 days after HFS at different intensities and 7 days after spared nerve injury (SNI). Scale bar, 100 μm. n = 3–4 mice/group, 3 slices/mouse, ***P < 0.001, compared with sham group, ###P < 0.001 vs. 20V HFS, one-way ANOVA with Tukey’s test. (D) The latencies to fall in rotarod test in different groups at 7 days after HFS at different intensities or SNI are shown. n = 5–12 mice for each group, ***P < 0.001, compared with sham group, one-way ANOVA with Tukey’s test. (E) Immunofluorescence of Iba1 (a marker for resident macrophage) and NIMP-R14 (a marker for neutrophil) in the stimulated sciatic nerve from different groups at 7days after HFS or SNI are shown (n = 3 mice/group, 2–3 sections/mouse). Scale bar: 50 μm. (F) Quantification of Iba1 expression in the sciatic nerve. ***P < 0.001, compared with sham group, ###P < 0.001 vs. 20V HFS, one-way ANOVA with Tukey’s test. (G, H) HFS at 10V and 20V significantly decreased 50% paw withdrawal threshold (PWT, G) to von Frey filaments and paw withdrawal latency (PWL, H) to radiant thermal stimuli, compared with the sham group or 1V HFS (n = 12 mice for 10V HFS group, n = 5–6 mice for other groups, **P < 0.01, ***P < 0.001 vs. sham group, two-way ANOVA with Fisher’s LSD test). (I) The decrease in mechanical thresholds induced by 10 V HFS was prevented by local application of 2% lidocaine (50 μl) at the stimulated sciatic nerve 15 min before HFS (n = 5–7 mice/group). ###P < 0.001 vs. HFS group, two-way ANOVA with Fisher LSD’s test. (J) Intrathecal injection of NMDA receptor antagonist D-AP5 (50 μg/ml, 5 μl) but not vehicle (Vehi, PBS) 30 min before HFS abolished HFS-induced mechanical hypersensitivity (n = 6–8 mice/group). ##P < 0.01, ###P < 0.001 vs. vehicle group, two-way ANOVA with Fisher LSD’s test.

Article Snippet: The tissue sections/cultured DRG neurons were first blocked with 5% donkey serum in 0.3% Triton X-100 (Sigma) for 60/30 min at room temperature (RT), and then incubated overnight at 4°C with a mixture of rabbit anti-ATF3 (1:500, sc-188, Santa Cruz Biotechnology, USA), mouse anti-CGRP antibody [4901] (1:2000, ab81887, Abcam, USA), isolectin B4-FITC (1:100, L2895, Sigma, USA), rabbit anti-GAP43 antibody (1:500, ab128005, Abcam, USA), rabbit anti-Substance P receptor antibody (1:500, AB5060, Millipore, USA.), rabbit anti-Iba1 (1:2000, 019–19741, Wako), goat anti-M-CSF antibody (1:500, AF416, R&D Systems, USA); rat anti-CD11b (1:500, 101202, Biolegend, USA); goat anti-Iba1 (1:500, ab5076, Abcam, USA) or rabbit anti-CSF1R (1:500, sc-692, Santa Cruz Biotechnology, USA).

Techniques: Software, Expressing, Immunofluorescence, Marker, Injection

Journal: Cell reports

Article Title: Microglia are Indispensable for Synaptic Plasticity in the Spinal Dorsal Horn and Chronic Pain

doi: 10.1016/j.celrep.2019.05.087

Figure Lengend Snippet: (A) HFS upregulated BDNF and CGRP levels in SDH, and the effects were prevented by intrathecal injection of anti-CSF1 (40.0 ng/μl) 30 min before HFS (n = 3–6 mice/group). ***P < 0.001 vs. control sham group. ##P < 0.01; ###P < 0.001 vs. control HFS group; $P < 0.05 vs. anti-CSF1 sham group, one-way ANOVA with Fisher’s LSD test. (B-D) In cultured spinal cord slices, CSF1 (1 μg/ml) markedly upregulated microglial markers (CD11b and Iba1) and CSF1R, and the effects were dose-dependently suppressed by p38 MAPK inhibitor SB203580 (10 μM) applied 1 hour prior to CSF1. Scale bar: 100 μm. n = 4–5 samples/group,* P < 0.05, ***P < 0.001 vs control group; ###P < 0.001 vs CSF1 group, one-way ANOVA with Tukey’s post-hoc test. (E-F) In culture spinal cord slices, treatment with CSF1 for 6 hours upregulated CSF1R, the phospho-p38 MAPK (p-p38) and BDNF, as well as increased BDNF in culture media. Pretreatment with p38 inhibitor SB203580 (10 μM) abolished the effects of CSF1. n = 3–5 samples/group, **P < 0.01, ***P < 0.001 vs control group; ##P < 0.01 vs CSF1 group, one-way ANOVA with Tukey’s post-hoc test. (G) Intrathecal CSF1 upregulated CGRP expression in BDNF+/+ mice but not in mice deficient of microglial BDNF (BDNF−/−), as measured 3 days after injection (n = 3–6 mice/group). ***P < 0.001 vs. BDNF+/+PBS group, ###P < 0.001 vs. BDNF+/+ CSF1 group, one-way ANOVA with Fisher’s LSD test. (H) The mechanical allodynia induced by intrathecal CSF1 was significantly attenuated in microglial BDNF deletion mice compared with control mice (n = 5–6 mice/group, ###P < 0.001 vs. BDNF+/+ CSF1 group), two-way ANOVA with Fisher’s LSD test.

Article Snippet: The tissue sections/cultured DRG neurons were first blocked with 5% donkey serum in 0.3% Triton X-100 (Sigma) for 60/30 min at room temperature (RT), and then incubated overnight at 4°C with a mixture of rabbit anti-ATF3 (1:500, sc-188, Santa Cruz Biotechnology, USA), mouse anti-CGRP antibody [4901] (1:2000, ab81887, Abcam, USA), isolectin B4-FITC (1:100, L2895, Sigma, USA), rabbit anti-GAP43 antibody (1:500, ab128005, Abcam, USA), rabbit anti-Substance P receptor antibody (1:500, AB5060, Millipore, USA.), rabbit anti-Iba1 (1:2000, 019–19741, Wako), goat anti-M-CSF antibody (1:500, AF416, R&D Systems, USA); rat anti-CD11b (1:500, 101202, Biolegend, USA); goat anti-Iba1 (1:500, ab5076, Abcam, USA) or rabbit anti-CSF1R (1:500, sc-692, Santa Cruz Biotechnology, USA).

Techniques: Injection, Cell Culture, Expressing

Journal: Cell reports

Article Title: Microglia are Indispensable for Synaptic Plasticity in the Spinal Dorsal Horn and Chronic Pain

doi: 10.1016/j.celrep.2019.05.087

Figure Lengend Snippet: (A) Western blots show the time course of Iba1 expression level after 10V HFS (n = 3 mice/ group). ***P< 0.001 vs. sham group, one-way ANOVA with Fisher’s LSD test. (B, C) Representative images of Iba1 immunostaining and the histogram show that the number of Iba1+ microglia were increased in the ipsilateral SDH at 1, 3 and 7 days after 10V HFS, and 7 days after SNI. Iba1+ cells were increased only in ipsilateral but not in contralateral SDH 7 days after HFS (7dI and 7dC). Scale bar: 50 μm. n = 3 mice/group, 2–3 sections/mouse. ***P < 0.001, ****P < 0.0001vs. sham group, ###P < 0.001 vs. HFS 7d group, one-way ANOVA with Fisher’s LSD test. (D) Experiment designs for control or microglial ablation (MG abl) at 3 or 21 days after vehicle (vehi, corn oil) diphtheria toxin treatment in CX3CR1CreER/+: R26iDTR/+mice. DT: diphtheria toxin, TM: Tamoxifen, Pain Beha: pain behavior test, EP: electrophysiology. In DT 3d HFS or DT 21d HFS group, DT was injected 3 weeks after TM and HFS was delivered at 3 or 21 days after last application of DT. In control group, vehicle (Vehi) but not DT was injected following TM injection. (E) The immunofluorescent staining showed that microglia were largely depleted at 3 days after DT treatment (DT 3d), and then fully repopulated within 21 days after DT treatment (DT 21d) in CX3CR1CreER/+: R26iDTR/+ mice. Boxed areas are magnified in the right panel. Scale bars: 100 μm (left) and 25 μm (right). (F) HFS was able to induce LTP at 21 days but not at 3 days after DT injection (n = 12 mice for DT 3 d group, n = 6–7 mice for other groups, two-way ANOVA with Tukey’s test). Representative traces of evoked C-fiber fEPSP are shown above before (i, grey line) and 3 h after 10V HFS (ii, colored line). Scale bars: x = 100 ms, y = 0.15 mV. The arrow indicates the time point at which HFS was delivered. (G) The input-output curves of fEPSP measured 7 d after HFS in mice treated with DT (for microglial ablation) and with vehicles are shown (n = 5 mice for sham group, n = 5 or 7 mice for MG ablation groups). (H, I) The increase in CGRP+ terminals induced by HFS was substantially attenuated in microglia ablation mice (n = 3 mice/group, 2–3 sections/mouse). Scale bar: 20 μm. ***P < 0.001 vs. control sham group; #P < 0.05, ##P < 0.01 vs. control HFS 3d or 7d group, one-way ANOVA with Fisher’s LSD test. (J) HFS induced mechanical allodynia in mice with microglia full repopulation (DT 21d), but not in mice with microglia ablation (DT 3d). n = 12 mice in MG ablation DT 3d group, n = 8 mice for other groups. **P < 0.01, ***P < 0.001 vs. Control HFS, two-way ANOVA with Fisher’s LSD test.

Article Snippet: The tissue sections/cultured DRG neurons were first blocked with 5% donkey serum in 0.3% Triton X-100 (Sigma) for 60/30 min at room temperature (RT), and then incubated overnight at 4°C with a mixture of rabbit anti-ATF3 (1:500, sc-188, Santa Cruz Biotechnology, USA), mouse anti-CGRP antibody [4901] (1:2000, ab81887, Abcam, USA), isolectin B4-FITC (1:100, L2895, Sigma, USA), rabbit anti-GAP43 antibody (1:500, ab128005, Abcam, USA), rabbit anti-Substance P receptor antibody (1:500, AB5060, Millipore, USA.), rabbit anti-Iba1 (1:2000, 019–19741, Wako), goat anti-M-CSF antibody (1:500, AF416, R&D Systems, USA); rat anti-CD11b (1:500, 101202, Biolegend, USA); goat anti-Iba1 (1:500, ab5076, Abcam, USA) or rabbit anti-CSF1R (1:500, sc-692, Santa Cruz Biotechnology, USA).

Techniques: Western Blot, Expressing, Immunostaining, Injection, Staining

Journal: Cell reports

Article Title: Microglia are Indispensable for Synaptic Plasticity in the Spinal Dorsal Horn and Chronic Pain

doi: 10.1016/j.celrep.2019.05.087

Figure Lengend Snippet: (A, B) The representative images and statistical analysis show the expression of CSF1 in the ipsilateral L4 DRG from 1 to 7 days after HFS and 7 days after SNI. The triple staining images showed the colocalization of CSF1, CGRP and ATF3. The arrowheads indicate that the injured DRG neurons (ATF3+) that strongly express CSF1 does not express CGRP. The arrows indicate the uninjured neurons (ATF3−) that weakly express CSF1 express CGRP following HFS or SNI. Scale bar: 50 μm. n = 3 mice/group, n = 2–3 images/mouse, *P < 0.05, ***P < 0.001 vs sham group; ###P < 0.001, compared with HFS 7d group, one-way ANOVA with Tukey’s post-hoc test. (C) Triple staining show CSF1 in SDH was co-localized with CGRP but not with IB4 at 3 days after HFS. Scale bar: 20 μm. (D) qRT-PCR analysis for CSF1 mRNA in the ipsilateral L4–5 DRGs from different groups (n = 7–12 mice/group). Values are presented as means ± SEM. *P < 0.05, **P < 0.01, ****P < 0.00001 vs. sham group; ###P < 0.001, compared with HFS 7d group, one-way ANOVA with Fisher’s LSD test. (E) Western blot and statistical analysis show that treatment of cultured DRG neurons with 40 mM KCl for 24 h increased CSF1 in culture media. n = 6 samples/group, **P < 0.01 vs Vehi group, unpaired Student’s t-test. (F, G) Immunostaining shows CSF1R in microglia was upregulated in the ipsilateral dorsal horn at 1 and 3 days after HFS (n = 3 mice/group, 2–3 sections/mouse). Scale bar: 50 μm. ***P < 0.001, compared with sham group, ###P < 0.001, compared with HFS 7d, one-way ANOVA with Tukey’s post-hoc test. (H) Western blots illustrate the increased expression of CSF1R and Iba1 protein levels at 3 days after 10V HFS, and the changes were blocked by intrathecal injection of CSF1 neutralizing antibody (anti-CSF1, 40 ng/μl, 5 μl) 30 min before HFS (n = 3–4 mice/group). **P < 0.01, ***P < 0.001 vs. sham group. ###P < 0.001 vs. control HFS group, one-way ANOVA with Fisher’s LSD test. (I) Local application of anti-CSF1 (40 ng/μl, 20 μl) onto the dorsal surface of spinal cord at recording segments 30 min before HFS did not affect baseline of C-fiber responses, but blocked late-phase of the spinal LTP (n = 5–6 mice/group). Original traces of C-fiber fEPSPs from different groups, recorded at the baseline (i), 30 min before (ii) and 3 h (iii) after HFS, are shown on the top. Scale bars: x = 100 ms, y = 0.2 mV. The arrow indicates the time point at which conditional stimulation was delivered. (J) Intrathecal injection of anti-CSF1 antibody 30 min before HFS prevented the development of mechanical hypersensitivity (n = 6 mice/group, ***P < 0.001 vs. vehicle group (Vehi), two-way ANOVA with Fisher’s LSD test.

Article Snippet: The tissue sections/cultured DRG neurons were first blocked with 5% donkey serum in 0.3% Triton X-100 (Sigma) for 60/30 min at room temperature (RT), and then incubated overnight at 4°C with a mixture of rabbit anti-ATF3 (1:500, sc-188, Santa Cruz Biotechnology, USA), mouse anti-CGRP antibody [4901] (1:2000, ab81887, Abcam, USA), isolectin B4-FITC (1:100, L2895, Sigma, USA), rabbit anti-GAP43 antibody (1:500, ab128005, Abcam, USA), rabbit anti-Substance P receptor antibody (1:500, AB5060, Millipore, USA.), rabbit anti-Iba1 (1:2000, 019–19741, Wako), goat anti-M-CSF antibody (1:500, AF416, R&D Systems, USA); rat anti-CD11b (1:500, 101202, Biolegend, USA); goat anti-Iba1 (1:500, ab5076, Abcam, USA) or rabbit anti-CSF1R (1:500, sc-692, Santa Cruz Biotechnology, USA).

Techniques: Expressing, Staining, Quantitative RT-PCR, Western Blot, Cell Culture, Immunostaining, Injection

Journal: Cell reports

Article Title: Microglia are Indispensable for Synaptic Plasticity in the Spinal Dorsal Horn and Chronic Pain

doi: 10.1016/j.celrep.2019.05.087

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The tissue sections/cultured DRG neurons were first blocked with 5% donkey serum in 0.3% Triton X-100 (Sigma) for 60/30 min at room temperature (RT), and then incubated overnight at 4°C with a mixture of rabbit anti-ATF3 (1:500, sc-188, Santa Cruz Biotechnology, USA), mouse anti-CGRP antibody [4901] (1:2000, ab81887, Abcam, USA), isolectin B4-FITC (1:100, L2895, Sigma, USA), rabbit anti-GAP43 antibody (1:500, ab128005, Abcam, USA), rabbit anti-Substance P receptor antibody (1:500, AB5060, Millipore, USA.), rabbit anti-Iba1 (1:2000, 019–19741, Wako), goat anti-M-CSF antibody (1:500, AF416, R&D Systems, USA); rat anti-CD11b (1:500, 101202, Biolegend, USA); goat anti-Iba1 (1:500, ab5076, Abcam, USA) or rabbit anti-CSF1R (1:500, sc-692, Santa Cruz Biotechnology, USA).

Techniques: Recombinant, Software, Microscopy

Journal: Oncology Letters

Article Title: Quercetin induces tongue squamous cell carcinoma cell apoptosis via the JNK activation-regulated ERK/GSK-3α/β-mediated mitochondria-dependent apoptotic signaling pathway

doi: 10.3892/ol.2022.13198

Figure Lengend Snippet: Effects of quercetin on cell morphology and viability in human tongue squamous cell carcinoma SAS cells. (A) Cells were treated with quercetin (30 and 50 µM) for 24 h. The morphological changes of cells were observed under an inverted phase-contrast microscope (magnification, ×400). (B) Cells were treated with quercetin (10–300 µM) for 24 h. Cell viability was determined using the MTT assay. (C-a) Apoptosis was determined by staining with the Annexin V-FITC apoptosis assay kit and analyzed using flow cytometry, and quantitative analysis of the (C-b) percentage of phosphatidylserine exposure on the outer cellular membrane and (C-c) early and late apoptotic cells (Annexin V + /PI − and Annexin V + /PI + ) was detected. (D) Cells were treated with quercetin (10–50 µM) for 24 h. Caspase-3 activity was examined by a Caspase-3 Activity Assay Kit. Cisplatin (10 µg/ml) was used as a positive control. In B-D, data are presented as the mean ± standard deviation of four independent experiments with triplicate determination. *P<0.05 vs. vehicle control.

Article Snippet: To assess quercetin-induced apoptosis and necrosis, flow cytometry was performed using the Annexin V-FITC-PI assay kit (BioVision, Inc.).

Techniques: Microscopy, MTT Assay, Staining, Apoptosis Assay, Flow Cytometry, Membrane, Activity Assay, Caspase-3 Activity Assay, Positive Control, Standard Deviation, Control

Journal:

Article Title: Genetic Susceptibility and Caspase Activation in Mouse and Human Macrophages Are Distinct for Legionella longbeachae and L. pneumophila ▿

doi: 10.1128/IAI.00025-07

Figure Lengend Snippet: Detection of caspase 3 activation by its cleavage of a fluorescent substrate. (A) Human U937 macrophages were infected with the L. longbeachae clinical isolate D4968 and L. pneumophila, and the number of CFU was monitored over 48 h. (B) Human U937 macrophages were infected with the L. longbeachae clinical isolate D4968, D4969, or D4973, L. pneumophila, or its dotA isogenic mutant. The caspase 3 activity to cleave the fluorescent substrate was measured at 6 h postinfection and expressed as arbitrary fluorescence units (AFU). The data are representative of three independent experiments, and the error bars represent standard deviations.

Article Snippet: At 6 h postinfection, the activity of caspase 3 in the infected U937 cells was measured by a fluorometric caspase 3 assay kit (BioVision, Inc., CA) as described previously ( 35 ).

Techniques: Activation Assay, Infection, Mutagenesis, Activity Assay, Fluorescence

Journal:

Article Title: Genetic Susceptibility and Caspase Activation in Mouse and Human Macrophages Are Distinct for Legionella longbeachae and L. pneumophila ▿

doi: 10.1128/IAI.00025-07

Figure Lengend Snippet: Single-cell analysis of caspase 3 activation in human macrophages. hMDMs were infected with the L. longbeachae clinical isolate D4968, L. pneumophila, or its dotA isogenic mutant at an MOI of 10, and caspase 3 activation was analyzed at 6 h and 24 h postinfection. Bacteria were detected by a specific antibody, while active caspase 3 was detected by an antiactive caspase 3 antibody. (A) Representative confocal microscopy images at several time points are shown. (B) Quantification of the percentage of cells with active caspase 3 was determined by analysis of 100 infected cells from three different coverslips in each experiment. There were equivalent numbers of bacteria per phagosome for both bacterial species. The data are representative of three independent experiments, and the error bars represent standard deviations.

Article Snippet: At 6 h postinfection, the activity of caspase 3 in the infected U937 cells was measured by a fluorometric caspase 3 assay kit (BioVision, Inc., CA) as described previously ( 35 ).

Techniques: Single-cell Analysis, Activation Assay, Infection, Mutagenesis, Confocal Microscopy

Journal:

Article Title: Genetic Susceptibility and Caspase Activation in Mouse and Human Macrophages Are Distinct for Legionella longbeachae and L. pneumophila ▿

doi: 10.1128/IAI.00025-07

Figure Lengend Snippet: L. longbeachae triggers low caspase 3 activation in murine macrophages. A/J and BALB/c mouse macrophages were infected with the L. longbeachae clinical isolate D4968 and ATCC 33462, and levels of caspase 3 activation (C3) and apoptosis (TUNEL [T]) were determined at 2, 8, and 18 h postinfection. L. pneumophila and its isogenic dotA mutant were used as positive and negative controls, respectively. The data are representative of three experiments.

Article Snippet: At 6 h postinfection, the activity of caspase 3 in the infected U937 cells was measured by a fluorometric caspase 3 assay kit (BioVision, Inc., CA) as described previously ( 35 ).

Techniques: Activation Assay, Infection, TUNEL Assay, Mutagenesis

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Antibody-Directed Glucocorticoid Targeting to CD163 in M2-type Macrophages Attenuates Fructose-Induced Liver Inflammatory Changes

doi: 10.1016/j.omtm.2016.11.004

Figure Lengend Snippet: CD163-Positive Liver Macrophages in Rats on the Standard Diet versus the HFr Diet (A and B) Anti-CD163 antibody staining of Kupffer cells/infiltrating macrophages using immunohistochemistry of fixed paraffin-embedded liver sections from rats fed the (A) STD or (B) HFr diet for 2 weeks. White scale bars represent 400 μm. (C and D) Fluorescence microscopic analysis of formalin-fixed cryosections of rat liver 1 hr after i.v. injection of (C) the anti-CD163-dexamethasone conjugate and (D) the control IgG-dexamethasone conjugate in rats on the STD. Anti-mouse IgG-Alexa Fluor 488 (green) was used for detection of the conjugates. Staining of the CD163-dexamethasone conjugate is in accordance with uptake in Kupffer cells (arrowheads). Scale bars represent 20 μm.

Article Snippet: A goat anti-rabbit Alexa Fluor 647 antibody (Abcam) and a goat anti-mouse IgG-Alexa Fluor 488 antibody (Abcam) were used as secondary antibodies for staining of the conjugates.

Techniques: Staining, Immunohistochemistry, Fluorescence, Injection

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Antibody-Directed Glucocorticoid Targeting to CD163 in M2-type Macrophages Attenuates Fructose-Induced Liver Inflammatory Changes

doi: 10.1016/j.omtm.2016.11.004

Figure Lengend Snippet: Targeting of Anti-CD163-Dexamethasone to Rat Kupffer Cells Confocal fluorescence microscopy analysis of liver cell suspensions after 30 min of incubation with (A–C) the anti-CD163-dexamethasone conjugate or (D–F) the IgG-dexamethasone conjugate. (A and D) Anti-mouse IgG-Alexa Fluor 488 (green) staining of CD163 antibody. (B and E) Anti-rabbit Alexa Fluor 647 (red) staining of dexamethasone. (C) shows the overlay of (A) and (B) and (F) shows the overlay of (D) and (E), with DAPI (light blue) nuclei staining showing co-location of anti-CD163 and dexamethasone. Scale bars represent 20 μm.

Article Snippet: A goat anti-rabbit Alexa Fluor 647 antibody (Abcam) and a goat anti-mouse IgG-Alexa Fluor 488 antibody (Abcam) were used as secondary antibodies for staining of the conjugates.

Techniques: Fluorescence, Microscopy, Incubation, Staining

Journal: The American Journal of Pathology

Article Title: Profiles of Cancer Stem Cell Subpopulations in Cholangiocarcinomas

doi: 10.1016/j.ajpath.2015.02.010

Figure Lengend Snippet: List of Antibodies Used for IHC and IF

Article Snippet: For all immunoreactions, negative controls (the primary antibody was replaced with preimmune serum) were also included. shows the details of antibodies used in the study. table ft1 table-wrap mode="anchored" t5 caption a7 Name Host/isotype Source Catalog# Dilution CD326/EpCAM Mouse IgG1 Santa Cruz Biotechnology (Dallas, TX) sc-59782 1:50 K7 (cytokeratin 7) Mouse IgG1 Dako M7018 1:100 K19 (cytokeratin 19) Mouse IgG1 Abcam (Cambridge, UK) ab87014 1:50 K19 (cytokeratin 19) Mouse IgG1 Dako M0888 1:100 HepPar-1 Mouse IgG1 Dako M7158 1:50 CD133/Prominin 1 Rabbit IgG Abnova (Taipei, Taiwan) {"type":"entrez-protein","attrs":{"text":"PAB12663","term_id":"1236625334","term_text":"PAB12663"}} PAB12663 1:100 CD133/PROM1 Mouse IgG1 OriGene (Unimed Scientifica, Rome, Italy) TA309943 1:50 CD90/Thy1 Rabbit IgG Abcam ab92574 1:100 CD13 Mouse IgG1 Novacastra Reagents (Leica Biosystems, Buffalo Grove, IL) NCL-CD13-304 1:100 LGR5 Goat IgG Santa Cruz Biotechnology SC-68580 1:50 Desmin Mouse IgG1 Dako M0760 1:100 Vimentin Mouse IgG1 Santa Cruz Biotechnology sc-32322 1:100 Nestin Mouse IgG1 Santa Cruz Biotechnology sc-23927 1:100 α-SMA Mouse IgG1 Dako M0851 1:50 S100A4 Rabbit IgG Dako A5114 1:100 SNAIL Rabbit IgG Santa Cruz Biotechnology sc-28199 1:50 TWIST Rabbit IgG Santa Cruz Biotechnology sc-15393 1:50 LGR5 Rabbit IgG OriGene TA301323 1:50 NCAM-PE Mouse IgG1 BD Pharmingen (Milan, Italy) 555.516 1:50 E-cadherin Mouse IgG1 Santa Cruz Biotechnology sc-21791 1:50 P-cadherin Rabbit IgG Santa Cruz Biotechnology sc-7893 1:50 GFAP Mouse IgG1 Dako M0761 1:50 CD163 Mouse IgG1 OriGene TA506382 1:50 CD31 Mouse IgG1 Dako M0823 1:50 CD13 Mouse IgG1 Abcam Ab7417 1:100 SDF1 Rabbit IgG Santa Cruz Biotechnology sc-28876 1:50 FAP Mouse IgG1 Santa Cruz Biotechnology sc-65398 1:50 Periostin Goat IgG Santa Cruz Biotechnology sc-49480 1:50 Periostin Rabbit IgG Santa Cruz Biotechnology sc-67233 1:50 CD90-FITC Human Miltenyi Biotec (Cologne, Germany) 130-095-403 1:10 CD326 (EPCAM)-FITC Human Miltenyi Biotec 130-080-301 1:10 Goat anti-rabbit FITC IgG Abcam ab-6717 1:400 Goat anti-rabbit TRITC IgG Abcam ab-6718 1:400 Goat anti-mouse FITC IgG Abcam ab-6785 1:400 Goat anti-mouse TRITC IgG Abcam ab-6786 1:400 Open in a separate window List of Antibodies Used for IHC and IF Sections were examined in a coded fashion by Leica Microsystems DM 4500 B Light and Fluorescence Microscopy (Leica Microsystems, Weltzlar, Germany) equipped with a Jenoptik ProgRes C10 Plus Videocam (Jenoptik, Jena, Germany).

Techniques: