Journal: Journal of Neuroinflammation

Article Title: Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

doi: 10.1186/1742-2094-9-149

Figure Lengend Snippet: List of PCR primers used in the study

Article Snippet: The membranes were blocked with 5% skim milk, and sequentially incubated with primary antibodies (rabbit polyclonal anti-mouse PAI-1 antibody, anti-LRP1 antibody (H-80; raised against the N-terminal extracellular domain of 515 kDa LRP1) (both Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-STAT1 antibody, rabbit polyclonal anti-phospho-STAT1 antibody (both Cell Signaling Technology, Beverly, MA, USA), monoclonal anti-mouse TLR2 antibody, monoclonal anti-mouse TLR6 antibody monoclonal anti-mouse TLR9 antibody (all Imgenex, San Diego, CA, USA), or monoclonal anti-α-tubulin clone B-5-1-2 mouse ascites fluid (Sigma)), and horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-mouse IgG (Amersham Biosciences) and anti-rabbit IgG (Cell Signaling Technology)), followed by ECL detection (Amersham Biosciences).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Neuroinflammation

Article Title: Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

doi: 10.1186/1742-2094-9-149

Figure Lengend Snippet: Plasminogen activator inhibitor type 1 (PAI-1) downregulated Toll-like receptor (TLR)2/6 expression and its signaling. (A) BV-2 microglial cells were treated with PAI-1 (100 ng/ml) for 5 hours. TLR2/TLR6 and dectin-1 gene expression was detected by reverse transcriptase PCR. β-actin was used as an internal control. (B) Alternatively, BV-2 microglial cells were treated with PAI-1 (100 ng/ml) for 24 hours. The levels of TLR2, TLR6, and TLR9 protein were then evaluated by western blotting analysis. α-tubulin was used as an internal control. Values indicate the results of densitometric analysis normalized to either β-actin or α-tubulin. (C) Primary microglia cultures were treated with mouse PAI-1 protein (100 ng/ml), lipopolysaccharide (LPS; 100 ng/ml), interferon (IFN)-γ; 50 U/ml), and lipoteichoic acid (LTA; 1 μg/ml) as indicated for 24 hours. (Upper panel) NO production was measured by a Griess reaction. (Lower panel) Cell viability was measured by 2,5-diphenyltetrazolium bromide (MTT) reduction assays, and the results expressed as the percentage of surviving cells over the control cells . Results are given as mean ± SD ( n = 3). * P < 0.01, NS = not significant.

Article Snippet: The membranes were blocked with 5% skim milk, and sequentially incubated with primary antibodies (rabbit polyclonal anti-mouse PAI-1 antibody, anti-LRP1 antibody (H-80; raised against the N-terminal extracellular domain of 515 kDa LRP1) (both Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-STAT1 antibody, rabbit polyclonal anti-phospho-STAT1 antibody (both Cell Signaling Technology, Beverly, MA, USA), monoclonal anti-mouse TLR2 antibody, monoclonal anti-mouse TLR6 antibody monoclonal anti-mouse TLR9 antibody (all Imgenex, San Diego, CA, USA), or monoclonal anti-α-tubulin clone B-5-1-2 mouse ascites fluid (Sigma)), and horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-mouse IgG (Amersham Biosciences) and anti-rabbit IgG (Cell Signaling Technology)), followed by ECL detection (Amersham Biosciences).

Techniques: Expressing, Gene Expression, Reverse Transcription, Control, Western Blot

Journal: Biology Open

Article Title: Human hepatocyte depletion in the presence of HIV-1 infection in dual reconstituted humanized mice

doi: 10.1242/bio.029785

Figure Lengend Snippet: HIV-1 infection affects immune cell distribution in blood, spleen and liver tissues. (A) Gating strategy for analyzing frequency human cells distribution for blood, spleen and liver tissues. The results are expressed as percentages of the total number of gated lymphocytes. The gating strategy was human CD45→CD3/CD19, CD3→CD4/CD8. (B) Representative dot plots for uninfected (HIV-1−, n= 7) and HIV-infected (HIV-1+, n= 9) dual humanized mice showing human immune cell distribution in the blood, spleen and liver of mice. mCD45 indicates mouse specific CD45 + cells. Numbers indicate the percentage of gated cells. (C) Frequency of human cells in peripheral blood, spleen and liver of dual reconstituted uninfected (squares, HIV-1−, n =7) and HIV-1 infected (triangles, HIV-1+; n =9) mice. Pre-infection (PRE) in blood panel indicates the frequency of human cells prior to HIV-1 infection. Results represent the mean±s.e.m. as well as individual values. * P <0.05, and ** P <0.005 by one-way ANOVA test between HIV-infected and uninfected mice. (D) Pearson correlation of CD4 + /CD8 + ratio (ratios of CD4 + to CD8 + cells at 5 weeks post-infection) with HIV-1 RNA copies/ml in peripheral blood ( n =8, R 2 =0.75, P <0.05) and (E) with albumin ALB (ratios of ALB concentration at 5-weeks post-infection to pre-infection ALB levels at 4 months after Hep transplantation) ( n =7, R 2 =0.72, P <0.05).

Article Snippet: Human-specific primer-probe sets ( PTPRC , Hs04189704_m1; CXCL10 , Hs01124252_g1; TLR9 , Hs00370913_s1; TLR3 , Hs00152933_m1; IL23A , Hs00413259_m1; TLR7 , Hs01933259_s1; TLR4 , Hs00370853_m1; TLR1 , Hs00413978_m1) and GAPDH (Hs99999905_m1) were purchased from Applied Biosystems.

Techniques: Infection, Concentration Assay, Transplantation Assay

Journal: PloS one

Article Title: Synergic activation of toll-like receptor (TLR) 2/6 and 9 in response to Ureaplasma parvum & urealyticum in human amniotic epithelial cells.

doi: 10.1371/journal.pone.0061199

Figure Lengend Snippet: Figure 2. TLR2/6 and TLR9-dependent cytokine secretion in response to Ureaplasma serovars. Human amniotic epithelial cells (A, B) were either not stimulated (white bar charts) or stimulated with Ureaplasma serovars for 2 hours. The cells were fixed and permeabilised, followed by antibody staining against the particular TLR molecule, and incubation with the appropriate secondary antibody conjugated to FITC. Fluorescence was detected using a FACSCalibur (BectonDickinson The data presented is the mean of three independent experiments. HEK-293 cells (B) transfected with TLR1, TLR2, TLR2/6, TLR4, TLR7 and TLR9 were either not incubated (white bar charts) or incubated with with Ureaplasma (16108 bacteria/ml to 16107 cells/ml) parvum SV3 (black barcharts), U. parvum SV14 (grey barcharts), U. urealyticum SV2 (stripped bar charts), or MBA (1 mg/ml) for 2 h. Control cultures were stimulated with known TLR2, TLR4, TLR7 or TLR9 ligands. The supernatants were harvested and assayed for IL-6 content using the Cytometric Bead Array (CBA) system (Becton Dickinson). Fluorescence was detected using a FACSCalibur (BectonDickinson). The data represents the mean 6 SD of three independent experiments. Asterisks indicate statistically significant (p,0.05) increase in expression (A,B) or IL-6 secretion (C) compared to corresponding unstimulated controls. doi:10.1371/journal.pone.0061199.g002

Article Snippet: TLR1, TLR2, TLR4, TLR6, TLR7, and TLR9 specific polyclonal antibodies were obtained from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Staining, Incubation, Fluorescence, Transfection, Bacteria, Control, Expressing

Journal: PloS one

Article Title: Synergic activation of toll-like receptor (TLR) 2/6 and 9 in response to Ureaplasma parvum & urealyticum in human amniotic epithelial cells.

doi: 10.1371/journal.pone.0061199

Figure Lengend Snippet: Figure 3. Inhibition of Ureaplasma activation of human amniotic epithelial cells by silencing TLR2 and TLR9. TLR expression was knocked down by siRNA and confirmed by western blotting (A). Following RNA interference, human amniotic epithelial cells were not stimulated (white bar chasrts), or stimulated with Ureaplasma (16108 bacteria/ml to 16107 cells/ml) parvum SV3 (black barcharts), U. parvum SV14 (grey barcharts), U. urealyticum SV2 (stripped bar charts) or MBA (1 mg/ml) for 2 h (B). The supernatants were harvested and assayed for cytokine secretion using the Cytometric Bead Array (CBA) system (Becton Dickinson). Fluorescence was detected using a FACSCalibur (BectonDickinson). The data represents the mean 6 SD of three independent experiments. Asterisks indicate statistically significant (p,0.05) decrease in IL-6 secretion compared to corresponding unsilenced controls. doi:10.1371/journal.pone.0061199.g003

Article Snippet: TLR1, TLR2, TLR4, TLR6, TLR7, and TLR9 specific polyclonal antibodies were obtained from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Inhibition, Activation Assay, Expressing, Western Blot, Bacteria, Fluorescence

Journal: PloS one

Article Title: Synergic activation of toll-like receptor (TLR) 2/6 and 9 in response to Ureaplasma parvum & urealyticum in human amniotic epithelial cells.

doi: 10.1371/journal.pone.0061199

Figure Lengend Snippet: Figure 6. Ureaplasma internalization recruits MyD88 in endosomes. Human amniotic epithelial cells were either not stimulated (top panels) or stimulated with either Ureaplasma SV3 (middle panels) or MBA (bottom panels) and imaged using a Zeiss 510 META confocal microscope. Intracellular MyD88 was stained using a polyclonal antibody directly labelled with FITC. TLR9 were labelled using anti-TLR9-TRITC. TOPRO was used to label the nucleus of the cells. Merged images showing extensive overlay of areas positive for MyD88 and TLR9 are seen as yellow (Scale Bar, 10 mm). doi:10.1371/journal.pone.0061199.g006

Article Snippet: TLR1, TLR2, TLR4, TLR6, TLR7, and TLR9 specific polyclonal antibodies were obtained from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Microscopy, Staining