Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inflammatory cytokines break down intrinsic immunological tolerance of human primary keratinocytes to cytosolic DNA.

doi: 10.4049/jimmunol.1302120

Figure Lengend Snippet: FIGURE 4. Assembly of the DNA-activated signalsome in kerati- nocytes is supported by cytokine treatment. (A) Human primary ker- atinocytes were treated with FITC-dsDNA (delivered with Lipofectamine [Lipo], 2 mg/ml), LL37 (5 mg/ml), or TNF-a (50 ng/ml), as indicated. The cells were fixed after 2 h, stained for IFI16, STING, and TBK1, and analyzed by confocal microscopy. White box indicates area displayed in zoom column. Scale bar, 10 mm. (B) The cells were treated as in (A), but fixed after 6 h of treatment for subsequent staining and analysis. Similar results were obtained in at least three independent experiments. (C) Ker- atinocytes were left untreated (UT) or stimulated with 50 ng/ml of TNF-a or IL-1b for 2 h. Cytoplasmic extracts were analyzed for IFI16 and b-actin by Western blotting.

Article Snippet: For Western blotting, the following specific Abs were used: IFI16 (1G7; Santa Cruz Biotechnology, Santa Cruz, CA), cGAS (C6orf150; Sigma-Aldrich), RCC1 (sc-1162; Santa Cruz Biotechnology), and b-actin (AC-15 HRP; Abcam). at M ichigan State U niversity on February 8, 2015 http://w w w .jim m unol.org/ D ow nloaded from

Techniques: Staining, Confocal Microscopy, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inflammatory cytokines break down intrinsic immunological tolerance of human primary keratinocytes to cytosolic DNA.

doi: 10.4049/jimmunol.1302120

Figure Lengend Snippet: FIGURE 5. DNA-driven gene expression in cyto- kine-treated keratinocytes is dependent on IFI16. (A) Human primary keratinocytes were transfected with control (Ctrl) and IFI16-specific siRNA. Total RNA was harvested 48 h later, and levels of IFI16 mRNA were measured by RT-qPCR. (B and C) Keratinocytes treated with siRNA for 48 h were stimulated with synthetic dsDNA (Lipofectamine [Lipo], 2 mg/ml) or TNF-a (50 ng/ml) as indicated for 6 h. Total RNAwas isolated, and mRNA levels of CCL20 and CXCL10 were quantified by RT-qPCR. The data were normal- ized to b-actin and are presented as mean fold in- duction of triplicate cultures 6 SD. Similar results were obtained in at least three independent experi- ments. *p , 0.05. NR, Normalized ratio.

Article Snippet: For Western blotting, the following specific Abs were used: IFI16 (1G7; Santa Cruz Biotechnology, Santa Cruz, CA), cGAS (C6orf150; Sigma-Aldrich), RCC1 (sc-1162; Santa Cruz Biotechnology), and b-actin (AC-15 HRP; Abcam). at M ichigan State U niversity on February 8, 2015 http://w w w .jim m unol.org/ D ow nloaded from

Techniques: Gene Expression, Transfection, Control, Quantitative RT-PCR, Isolation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inflammatory cytokines break down intrinsic immunological tolerance of human primary keratinocytes to cytosolic DNA.

doi: 10.4049/jimmunol.1302120

Figure Lengend Snippet: FIGURE 6. IFI16 is upregulated in psoriasis lesions and exhibits a cellular localization pattern similar to keratinocytes receiving dual stimulation with DNA and inflammatory cytokines. (A) Tissue sections of punch biopsies from nonlesional and lesional psoriatic skin were stained for IFI16 and DNA (DAPI) and analyzed by confocal microscopy. Arrows indicate cytosolic IFI16 foci. Scale bar, 100 mm. (B) RNA was isolated from nonlesional (NLS) and lesional psoriatic skin (LS), and levels of IFI16 mRNA were quantified by RT- qPCR. Data shown as means of normalized values relative to nonlesional psoriasis skin 6 SD. n = 9. (C) Tissue section of punch biopsies from lesional psoriatic skin stained for DNA (DAPI) and analyzed by confocal microscopy. Arrows indicate DAPI staining (DNA) in the cytosol. Scale bar, 10 mm. *p , 0.05. DIC, Dif- ferential interference contrast; NR, Normalized ratio.

Article Snippet: For Western blotting, the following specific Abs were used: IFI16 (1G7; Santa Cruz Biotechnology, Santa Cruz, CA), cGAS (C6orf150; Sigma-Aldrich), RCC1 (sc-1162; Santa Cruz Biotechnology), and b-actin (AC-15 HRP; Abcam). at M ichigan State U niversity on February 8, 2015 http://w w w .jim m unol.org/ D ow nloaded from

Techniques: Staining, Confocal Microscopy, Isolation, Quantitative RT-PCR

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Immunohistochemical and Biochemical Expression Patterns of TTF-1, RAGE, GLUT-1 and SOX2 in HCV-Associated Hepatocellular Carcinomas

doi: 10.22034/APJCP.2018.19.1.219

Figure Lengend Snippet: Difference in Tissue Expression Scores of Studied Markers in Examined Lesions

Article Snippet: 4) SOX2 Antibody (NBP2-29623) (Novus Biologicals, USA) at an optimal dilution of 1:100 in PBS.

Techniques: Expressing

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Immunohistochemical and Biochemical Expression Patterns of TTF-1, RAGE, GLUT-1 and SOX2 in HCV-Associated Hepatocellular Carcinomas

doi: 10.22034/APJCP.2018.19.1.219

Figure Lengend Snippet: Difference in Markers Expression Scores in Different Ggrades of Hepatitis Activity and Stages of Fibrosis

Article Snippet: 4) SOX2 Antibody (NBP2-29623) (Novus Biologicals, USA) at an optimal dilution of 1:100 in PBS.

Techniques: Expressing, Activity Assay

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Immunohistochemical and Biochemical Expression Patterns of TTF-1, RAGE, GLUT-1 and SOX2 in HCV-Associated Hepatocellular Carcinomas

doi: 10.22034/APJCP.2018.19.1.219

Figure Lengend Snippet: Relation between the Expression of Tissue Markers and the Grades of HCC

Article Snippet: 4) SOX2 Antibody (NBP2-29623) (Novus Biologicals, USA) at an optimal dilution of 1:100 in PBS.

Techniques: Expressing

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Immunohistochemical and Biochemical Expression Patterns of TTF-1, RAGE, GLUT-1 and SOX2 in HCV-Associated Hepatocellular Carcinomas

doi: 10.22034/APJCP.2018.19.1.219

Figure Lengend Snippet: The Correlation between Serum TTF-1, RAGE, GLUT-1 and SOX-2 Genes Expression in Relation to Tumor Grade and Stage of Liver Fibrosis

Article Snippet: 4) SOX2 Antibody (NBP2-29623) (Novus Biologicals, USA) at an optimal dilution of 1:100 in PBS.

Techniques: Expressing

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Immunohistochemical and Biochemical Expression Patterns of TTF-1, RAGE, GLUT-1 and SOX2 in HCV-Associated Hepatocellular Carcinomas

doi: 10.22034/APJCP.2018.19.1.219

Figure Lengend Snippet: The Validation of SOX-2, GLUT-1, RAGE and TTF-1 Genes as a Diagnostic Biomarker in the Serum

Article Snippet: 4) SOX2 Antibody (NBP2-29623) (Novus Biologicals, USA) at an optimal dilution of 1:100 in PBS.

Techniques: Biomarker Discovery, Diagnostic Assay

Journal: The Journal of Neuroscience

Article Title: Aspirin Induces Lysosomal Biogenesis and Attenuates Amyloid Plaque Pathology in a Mouse Model of Alzheimer's Disease via PPARα

doi: 10.1523/JNEUROSCI.0054-18.2018

Figure Lengend Snippet: List of antibodies used in this study

Article Snippet: Mouse monoclonal (4G8) , Anaspec , SIG-39220-100 , WB/1:1000.

Techniques:

Journal: The Journal of Neuroscience

Article Title: Aspirin Induces Lysosomal Biogenesis and Attenuates Amyloid Plaque Pathology in a Mouse Model of Alzheimer's Disease via PPARα

doi: 10.1523/JNEUROSCI.0054-18.2018

Figure Lengend Snippet: Aspirin treatment reduces Aβ burden in the hippocampus of a 5XFAD mouse model of AD. A, Six- to 7-month-old 5XFAD mice (n = 6 per group) were treated orally with aspirin (2 mg/ kg body weight/d) or vehicle (0.1% methylcellulose) for 1 month and subsequently analyzed for Aβ levels in the hippocampal homogenates by immunoblotting using the 4G8 monoclonal antibody. β-Tubulin was used as the loading control. B, Densitometric analysis representing mean ± SEM for Aβ levels relative to non-Tg controls. C, Hippocampal sections demonstrating Aβ plaque pathology in transgenic 5XFAD mice in the CA1 region of hippocampus. D, Hhippocampal sections were double labeled using thio-S and Aβ 6E10 antibody. E–G, The thio-S-positive plaques in the hippocampus were further characterized for total area faction (thio-S positive area as a percentage of total hippocampal area; E), plaque count (F), and average plaque size (G). Data are shown as mean ± SEM. Quantification of thio-S-positive plaques were performed using ImageJ. H, Diaminobenzidine staining of hippocampal sections was performed using Aβ 6E10 antibody. In all cases, one-way ANOVA followed by Tukey's multiple-comparisons test was used for statistical analysis; *p < 0.05, **p < 0.001.

Article Snippet: Mouse monoclonal (4G8) , Anaspec , SIG-39220-100 , WB/1:1000.

Techniques: Western Blot, Transgenic Assay, Labeling, Staining

Journal:

Article Title: CX3CR1 Deficiency Alters Microglial Activation and Reduces Beta-Amyloid Deposition in Two Alzheimer's Disease Mouse Models

doi: 10.2353/ajpath.2010.100265

Figure Lengend Snippet: Reduced plaque-associated microglia in the APPPS1 mouse model of AD with CX3CR1 deficiency. Brain sections (30 μm) from APPPS1;Cx3cr1+/+ (A–C), APPPS1;Cx3cr1+/− (D–F), and APPPS1;Cx3cr1−/− (G–I) mice at 4 months of age were immunostained with monoclonal Aβ antibody 4G8 (red; A, D, and G), with an antibody against the pan-microglial marker Iba1 (green; B, E, and H), and counterstained with the nuclear TO-PRO-3 dye (blue, C, F, and I). Confocal microscopy was used to obtain maximum projections reconstructed from Z-stacks spanning 20–30 μm in depth. As expected, APPPS1;Cx3cr1+/+ controls exhibit extensive accumulation of Iba1-positive microglia around senile plaques (A–C). However, age-matched APPPS1;Cx3cr1+/− (D–F) and APPPS1;Cx3cr1−/− animals (G–I) exhibit gene dose-dependent reduction in the number of Iba1-positive microglia surrounding the senile plaques. The number of Iba1-positive microglia associated with senile plaques in the three genotypes was quantified in three nonadjacent sections from each of the four animals per genotype. APPPS1 mice with either one or two copies of Cx3cr1 loss-of-function alleles exhibited a statistically significant reduction in microglia surrounding both large (>1000 μm2; J), medium (>500 μm2, <1000 μm2; K), and small (<500 μm2; L) Aβ deposits when compared to age-matched APPPS1;Cx3cr1+/+ controls (*P < 0.05; **P < 0.001, respectively). The APPPS1;Cx3cr1+/− and APPPS1;Cx3cr1−/− genotypes exhibited a statistically significant difference in microglial accumulation around medium Aβ deposits (P < 0.05), suggesting a gene dose-dependent effect. n = 35 for all analyses. Scale bar = 25 μm.

Article Snippet: For double fluorescent immunohistochemistry, sections were rinsed in PBST, pretreated with sodium citrate buffer to quench microglial eGFP, cooled for 30 minutes at room temperature, blocked for one hour at room temperature, and incubated overnight at 4°C in blocking buffer containing anti-Iba1 rabbit polyclonal antibody (diluted 1:1000; Wako, Richmond, VA) and the anti-Aβ mouse monoclonal antibody 4G8 (diluted 1:500; Signet Labs).

Techniques: Marker, Confocal Microscopy