Journal: Protein & Cell

Article Title: Single-cell transcriptomic Atlas of aging macaque ocular outflow tissues

doi: 10.1093/procel/pwad067

Figure Lengend Snippet: Knockdown of APOE partially rescues TMCs degeneration with aging. Functional enrichment analyses in biological process, cellular component, and molecular function of cluster 0. Schematic diagram illustrating possible mechanisms of APOE in trabecular meshwork degeneration with aging. TMCs were transfected with 50 nmol/L APOE siRNA or negative control (NC) siRNA for 24 h, followed by stimulation with H 2 O 2 for 2 h and replacing fresh medium for more than 24 h. Transwell assay was used to investigate the migration ability of TMCs. (D) Quantification of cell migration in (C). (E) Western blot was performed to detect the protein levels of extracellular matrix (ECM) components including fibronectin, laminin, CD44, and α-SMA under different cell treatments. (F) Quantification of relative protein levels in (E). (G) Western blot was performed to detect the key molecular levels of PI3K-AKT pathway and the downstream caspase 3/9 under different cell treatments. (H) Quantification of relative protein levels in (G). (I) The apoptosis rate of TMCs in four groups was measured by flow cytometry. (J) Quantification of apoptotic proportion in all cells from (I). Data are presented as mean ± SEM values; * P < 0.05, ** P < 0.01, *** P < 0.001; n = 3. ns: no significance.

Article Snippet: Antibodies against CLU (12289-1-AP), VEGFA (19003-1-AP), P21 (10355-1-AP), laminin (23498-1-AP), CD44 (15675-1-AP), PI3K (60225-1-Ig), AKT (10176-2-AP), p-AKT (Ser473, 66444-1-Ig), caspase 3 (19677-1-AP) and caspase 9 (10380-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Knockdown, Functional Assay, Transfection, Negative Control, Transwell Assay, Migration, Western Blot, Flow Cytometry

Journal: Journal of Neuroinflammation

Article Title: Anti-tat Hutat2:Fc mediated protection against tat-induced neurotoxicity and HIV-1 replication in human monocyte-derived macrophages

doi: 10.1186/s12974-014-0195-2

Figure Lengend Snippet: Evaluation of the biological binding function of Hutat2:Fc and protective effects of Hutat2:Fc against HIV-1 Tat 86 -mediated toxicity in HTB-11 cells. (A) Specific binding of Hutat2:Fc to HIV-1 Tat. HIV-1 Tat 86 (Clade B) loaded nitrocellular membranes (NCM) were incubated with cell culture supernatants collected from HR-Hutat2-transduced HTB-11 (HTB-Hutat2), U937 (U937-Hutat2), or hMDM (hMDM-Hutat2) at 4°C overnight followed by incubation with rabbit anti-human IgG (H+L) and goat anti-rabbit IgG HRP conjugated antibodies. Specific binding was visualized by the color deposition on the NCM. The Tat 86 -loaded membrane incubated with rabbit anti-Tat serum served as a positive control (Pos Ctl) while incubated with cell culture supernatant from HR-A3H5 transduced HTB-11 served as a negative control (HTB-A3H5). The NCM loaded with Tat dilution buffer was used as a blank control (BLK Ctl). (B) Functional antagonization of Hutat2:Fc against HIV-1 Tat 86 -induced toxicity in HTB-11 cells by an MTT assay. The OD 570 value of untreated HTB-11 cells was arbitrarily defined as 100% cell viability. The relative cell viability (%) was expressed as a percentage relative to the untreated control cells. The cell viability was significantly higher for the cells treated with the conditioned mediums from transduced cells releasing Hutat:Fc when compared to the cultures that received Tat 86 (500 nM) alone (* P <0.01 for HTB-Hutat2 medium; # P <0.05 for U937-Hutat2 medium, and hMDM-Hutat2 medium). (C) Protection of HR-Hutat2 transduction against Tat 86 -induced toxicity by an MTT assay. No significant difference of cell viability was detected between normal and vector HR-Hutat2 transduced HTB-11 cells (HTB-Hutat2) ( P >0.05). However, the cell viability of HTB-11 transduced with the vector HR-Hutat2 was significantly higher than that of HTB-A3H5 in the presence of HIV-1 Tat 86 (500 nM) (* P <0.01). All experiments were performed in quadruplicate. Error bars denote the s.e.m.

Article Snippet: After washing three times with TBST, the plate was incubated with diluted Hutat2:Fc containing supernatant samples for 1 hour and then incubated with a goat anti-human IgG Fc biotin-conjugated detection antibody (Rockland) for 1 hour.

Techniques: Binding Assay, Incubation, Cell Culture, Membrane, Positive Control, Negative Control, Control, Functional Assay, MTT Assay, Transduction, Plasmid Preparation

Journal: PLoS ONE

Article Title: Local Administration of Soluble CD40:Fc to the Salivary Glands of Non-Obese Diabetic Mice Does Not Ameliorate Autoimmune Inflammation

doi: 10.1371/journal.pone.0051375

Figure Lengend Snippet: HEK293 cells were transduced with AAV2-CD40:Fc or were mock transduced. The fusion protein was detected by anti murine-CD40 (left panel) and anti-human-Fc (right) and showed a band ∼90 kD (size of non-reduced dimer) and a ∼45 kD (reduced monomer) band.

Article Snippet: The blot was blocked with 5% milk powder in TBST buffer and incubated with biotin labeled anti-murine CD40 (R&D systems, MAB4401), followed by streptavidin-HRP (R&D systems) or anti human IgG-HRP (Genetex) and developed with ECL reagent (GE Heatlhcare, Buckinghamshire, UK).

Techniques: Transduction

Journal: PLoS ONE

Article Title: Local Administration of Soluble CD40:Fc to the Salivary Glands of Non-Obese Diabetic Mice Does Not Ameliorate Autoimmune Inflammation

doi: 10.1371/journal.pone.0051375

Figure Lengend Snippet: Microarray analysis was performed on SG of CD40:Fc treated and control mice (n = 4 each, treatment at 10 weeks, end of study at 20 weeks) and gene changes greater than 2 fold were used in the analysis and compared for biological functional pathways or biomarkers by an Ingenuity Pathway analysis (IPA). ( A ) The pathway of NF-κB and TGF-β was affected in CD40:Fc treated mice. Green colored shapes indicates upregulated genes, red colored shapes downregulated; gray colored shapes genes in the pathway that were not differentially expressed. Color intensity reflects the degree of differential expression. Triangles represent phosphatases; horizontal diamond, peptidases; vertical diamond, enzymes; horizontal oval, transcription factors; vertical oval, transmembrane receptors; trapezoid, transporters; circle, other type of protein. Dotted lines indicate an indirect interaction; solid line, direct interaction, solid arrow head indicates “acts on”. ( B ) Metacore analysis of pathways linking differentially expressed genes to immune pathways. The differentially expressed genes are indicated along with the direction of change in expression. ( C ) Quantitative-PCR of selected genes shows agreement with microarray results. The results obtained using the microarray platform were validated by examining the correlation between the expression levels in the microarray and qPCR results obtained for a subset of genes.

Article Snippet: The blot was blocked with 5% milk powder in TBST buffer and incubated with biotin labeled anti-murine CD40 (R&D systems, MAB4401), followed by streptavidin-HRP (R&D systems) or anti human IgG-HRP (Genetex) and developed with ECL reagent (GE Heatlhcare, Buckinghamshire, UK).

Techniques: Microarray, Control, Functional Assay, Quantitative Proteomics, Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Local Administration of Soluble CD40:Fc to the Salivary Glands of Non-Obese Diabetic Mice Does Not Ameliorate Autoimmune Inflammation

doi: 10.1371/journal.pone.0051375

Figure Lengend Snippet: Up or down regulated genes in salivary glands of CD40:Fc treated mice versus control mice.

Article Snippet: The blot was blocked with 5% milk powder in TBST buffer and incubated with biotin labeled anti-murine CD40 (R&D systems, MAB4401), followed by streptavidin-HRP (R&D systems) or anti human IgG-HRP (Genetex) and developed with ECL reagent (GE Heatlhcare, Buckinghamshire, UK).

Techniques: Control

Journal: PLoS ONE

Article Title: Local Administration of Soluble CD40:Fc to the Salivary Glands of Non-Obese Diabetic Mice Does Not Ameliorate Autoimmune Inflammation

doi: 10.1371/journal.pone.0051375

Figure Lengend Snippet: FS was determined for each group of CD40:Fc treated mice and compared with control mice (n = 8–10 mice per group). Shown is the average FS per group. Error bar = standard deviation (SD), i.m. = intramuscular.

Article Snippet: The blot was blocked with 5% milk powder in TBST buffer and incubated with biotin labeled anti-murine CD40 (R&D systems, MAB4401), followed by streptavidin-HRP (R&D systems) or anti human IgG-HRP (Genetex) and developed with ECL reagent (GE Heatlhcare, Buckinghamshire, UK).

Techniques: Control, Standard Deviation

Journal: Arthritis and rheumatism

Article Title: Monocyte chemoattractant protein 1 released from glycosaminoglycans mediates its profibrotic effects in systemic sclerosis via the release of interleukin-4 from T cells.

doi: 10.1002/art.21497

Figure Lengend Snippet: Figure 1. CCR2 is not expressed by dermal fibroblasts from patients with systemic sclerosis (SSc). Cultured fibroblasts (n 6) were stained with mouse anti-human CCR2 IgG2B biotin-labeled antibodies as primary antibodies and phycoerythrin (PE)–conjugated streptavidin antibodies as secondary antibodies and analyzed by fluorescence-activated cell sorting. Samples incubated with IgG2B isotype antibodies and PE-conjugated streptavidin antibodies were used as controls. a and b, CCR2 was not detectable in SSc dermal fibroblasts. c and d, U937 cells, used as a positive control, showed strong expression of CCR2.

Article Snippet: For staining, 1 105 fibroblasts were incubated with 5 l of mouse anti-human CCR2 IgG2B biotin-labeled antibodies at a concentration of 50 ng/ l (R&D Systems) or with the same amounts of IgG2B isotype antibodies (Dako, Hamburg, Germany) for 45 minutes at 4°C in the dark.

Techniques: Cell Culture, Staining, Labeling, Fluorescence, FACS, Incubation, Positive Control, Expressing

Journal: Arthritis and rheumatism

Article Title: Monocyte chemoattractant protein 1 released from glycosaminoglycans mediates its profibrotic effects in systemic sclerosis via the release of interleukin-4 from T cells.

doi: 10.1002/art.21497

Figure Lengend Snippet: Figure 6. Role of monocyte chemoattractant protein 1 (MCP-1) in the development of fibrosis. MCP-1 does not have direct effects on dermal fibroblasts due to the lack of functional MCP-1 receptors. Instead, MCP-1 favors the differentiation of interleukin-4 (IL-4)– producing T cells. Soluble IL-4 in turn induces the synthesis of collagens in resident dermal fibroblasts via binding to IL-4 receptor. In this model, glycosaminoglycans, the levels of which are increased in fibrotic skin, bind MCP-1 via ionic–ionic interactions and act as a local reservoir for MCP-1, further enhancing its profibrotic effects.

Article Snippet: For staining, 1 105 fibroblasts were incubated with 5 l of mouse anti-human CCR2 IgG2B biotin-labeled antibodies at a concentration of 50 ng/ l (R&D Systems) or with the same amounts of IgG2B isotype antibodies (Dako, Hamburg, Germany) for 45 minutes at 4°C in the dark.

Techniques: Functional Assay, Binding Assay

Journal: Journal of neuroinflammation

Article Title: Anti-tat Hutat2:Fc mediated protection against tat-induced neurotoxicity and HIV-1 replication in human monocyte-derived macrophages.

doi: 10.1186/s12974-014-0195-2

Figure Lengend Snippet: Figure 3 Evaluation of the biological binding function of Hutat2:Fc and protective effects of Hutat2:Fc against HIV-1 Tat86-mediated toxicity in HTB-11 cells. (A) Specific binding of Hutat2:Fc to HIV-1 Tat. HIV-1 Tat86 (Clade B) loaded nitrocellular membranes (NCM) were incubated with cell culture supernatants collected from HR-Hutat2-transduced HTB-11 (HTB-Hutat2), U937 (U937-Hutat2), or hMDM (hMDM-Hutat2) at 4°C overnight followed by incubation with rabbit anti-human IgG(H+L) and goat anti-rabbit IgG HRP conjugated antibodies. Specific binding was visualized by the color deposition on the NCM. The Tat86-loaded membrane incubated with rabbit anti-Tat serum served as a positive control (Pos Ctl) while incubated with cell culture supernatant from HR-A3H5 transduced HTB-11 served as a negative control (HTB-A3H5). The NCM loaded with Tat dilution buffer was used as a blank control (BLK Ctl). (B) Functional antagonization of Hutat2:Fc against HIV-1 Tat86-induced toxicity in HTB-11 cells by an MTT assay. The OD570 value of untreated HTB-11 cells was arbitrarily defined as 100% cell viability. The relative cell viability (%) was expressed as a percentage relative to the untreated control cells. The cell viability was significantly higher for the cells treated with the conditioned mediums from transduced cells releasing Hutat:Fc when compared to the cultures that received Tat86 (500 nM) alone (*P <0.01 for HTB-Hutat2 medium; #P <0.05 for U937-Hutat2 medium, and hMDM-Hutat2 medium). (C) Protection of HR-Hutat2 transduction against Tat86-induced toxicity by an MTT assay. No significant difference of cell viability was detected between normal and vector HR-Hutat2 transduced HTB-11 cells (HTB-Hutat2) (P >0.05). However, the cell viability of HTB-11 transduced with the vector HR-Hutat2 was significantly higher than that of HTB-A3H5 in the presence of HIV-1 Tat86 (500 nM) (*P <0.01). All experiments were performed in quadruplicate. Error bars denote the s.e.m.

Article Snippet: After washing three times with TBST, the plate was incubated with diluted Hutat2:Fc containing supernatant samples for 1 hour and then incubated with a goat antihuman IgG Fc biotin-conjugated detection antibody (Rockland) for 1 hour.

Techniques: Binding Assay, Incubation, Cell Culture, Membrane, Positive Control, Negative Control, Control, Functional Assay, MTT Assay, Transduction, Plasmid Preparation