Journal: PLoS Pathogens

Article Title: DAMP Molecule S100A9 Acts as a Molecular Pattern to Enhance Inflammation during Influenza A Virus Infection: Role of DDX21-TRIF-TLR4-MyD88 Pathway

doi: 10.1371/journal.ppat.1003848

Figure Lengend Snippet: (A) Mouse J774A.1 macrophages were infected with IAV (2 MOI) in the presence of either control IgG (IgG) or anti-S100A9 blocking (neutralizing) antibody (S100A9 Ab). At indicated post-infection time-periods the medium supernatant was collected to assess levels of mouse IL-6 by ELISA. ( B ) Primary bone marrow derived macrophages (BMDM) isolated from wild-type (WT) and S100A9 knockout (KO) mice were infected with IAV (2 MOI). The medium supernatant was collected to assess levels of mouse IL-6 by ELISA. ( C ) WT and S100A9 KO BMDM were infected with IAV (2 MOI). At the indicated post-infection time-period, medium supernatant was collected to assess levels of mouse TNF-α(TNF) by ELISA. ( D ) S100A9 KO BMDMs were infected with IAV (2 MOI) in the presence of purified recombinant mouse S100A9 protein (5 µg/ml). Medium supernatant was collected from infected cells to assess levels of mouse TNF and IL-6 by ELISA. Vehicle control cells (veh) were incubated with HBSS buffer. The values represent the mean ± standard deviation from three independent experiments performed in triplicate. *p<0.05 using a Student's t test.

Article Snippet: The plates were washed three times with PBST and incubated with either goat anti-mouse IgG (300 ng/well) (R&D) (for mouse S100A9) or mouse anti-human IgG (50 ng/well) (R&D) (for human S100A9) in PBST+0.1% BSA for 2 h at room temperature.

Techniques: Infection, Blocking Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Isolation, Knock-Out, Purification, Recombinant, Incubation, Standard Deviation

Journal: PLoS Pathogens

Article Title: DAMP Molecule S100A9 Acts as a Molecular Pattern to Enhance Inflammation during Influenza A Virus Infection: Role of DDX21-TRIF-TLR4-MyD88 Pathway

doi: 10.1371/journal.ppat.1003848

Figure Lengend Snippet: ( A ) Mouse J774A.1 macrophages were incubated with purified recombinant mouse S100A9 protein (5 µg/ml) for 48 h and 72 h. The apoptotic state of these cells was examined by FACS analysis of annexin V and PI stained cells. Apoptosis rate (% apoptosis) was calculated based on number of annexin V positive/PI negative cells (denoting early apoptosis)+number of annexin V positive/PI positive cells (denoting late apoptosis)/total number of cells. ( B ) Mouse alveolar macrophage MH-S cell-line was incubated with purified S100A9 protein (5 µg/ml) for 48 h and 72 h. The apoptotic status was determined as described in (A). ( C ) Mouse J774A.1 macrophages were infected with IAV (2 MOI) in the presence of either control IgG (IgG) or anti-S100A9 blocking (neutralizing) antibody (S100A9 Ab). At 48 h post-infection, the apoptotic state of these cells was determined as described in (A). The values (i.e. annexin V and PI staining quantified by FACS) represents mean ± standard deviation from three independent experiments, *p<0.05 by Student's t test. Veh; cells incubated with HBSS buffer (vehicle control).

Article Snippet: The plates were washed three times with PBST and incubated with either goat anti-mouse IgG (300 ng/well) (R&D) (for mouse S100A9) or mouse anti-human IgG (50 ng/well) (R&D) (for human S100A9) in PBST+0.1% BSA for 2 h at room temperature.

Techniques: Incubation, Purification, Recombinant, Staining, Infection, Blocking Assay, Standard Deviation

Journal: PLoS Pathogens

Article Title: DAMP Molecule S100A9 Acts as a Molecular Pattern to Enhance Inflammation during Influenza A Virus Infection: Role of DDX21-TRIF-TLR4-MyD88 Pathway

doi: 10.1371/journal.ppat.1003848

Figure Lengend Snippet: ( A ) Survival of IAV infected (1×10 5 pfu/mouse via intra-tracheal route) mice administered with either control IgG (IgG) or anti-S100A9 blocking (neutralizing) antibody (S100A9 Ab) (24 h prior to IAV inoculation, 2 mg of antibody/mouse administered via i.p route). The data represents values from two independent experiments performed with 5 mice/group for each experiment (total 10 mice/group from two experiments); *p = 0.03. ( B ) Hematoxylin and eosin (H&E) staining of lung sections from mock infected or IAV infected mice (3×10 4 pfu/mouse via intra-tracheal route) administered with either control IgG (IgG) or S100A9 Ab (24 h prior to IAV inoculation, 2 mg of antibody/mouse was administered via i.p route). Magnification, ×10. ( C ) Mice were administered with purified recombinant mouse S100A9 protein (15 µg/mouse) via intra-tracheal route. At 8 h post-administration, levels of mouse TNF-α in the lung was assessed by performing ELISA analysis with lung homogenate. ( D ) Lung homogenate prepared from mock infected and IAV infected (2×10 4 pfu/mouse via intra-tracheal route) mice administered with either control IgG (IgG) or anti-S100A9 blocking (neutralizing) antibody (S100A9 Ab) (24 h prior to IAV inoculation, 2 mg of antibody/mouse administered via i.p route) were subjected to ELISA analysis to determine levels of mouse TNF-α in the lung. ( E ) For ex-vivo experiment, broncho-alveolar lavage fluid (BALF) was collected (at 3 d post-infection) from IAV infected mice (2×10 4 pfu/mouse via intra-tracheal route) administered with either control IgG (IgG) or anti-S100A9 blocking (neutralizing) antibody (S100A9 Ab) (24 h prior to IAV inoculation, 2 mg of antibody/mouse administered via i.p route). The BALF cells were isolated and plated in 48-well plate. After 2 h and 4 h, the medium supernatant was analyzed for mouse TNF-α (TNF) and mouse IL-6 by ELISA. Values shown in (C), (D) and (E) represent the mean ± standard deviation from three independent experiments performed in triplicate. *p<0.05 using a Student's t test. Veh; HBSS buffer diluted in PBS (vehicle control).

Article Snippet: The plates were washed three times with PBST and incubated with either goat anti-mouse IgG (300 ng/well) (R&D) (for mouse S100A9) or mouse anti-human IgG (50 ng/well) (R&D) (for human S100A9) in PBST+0.1% BSA for 2 h at room temperature.

Techniques: Infection, Blocking Assay, Staining, Purification, Recombinant, Enzyme-linked Immunosorbent Assay, Ex Vivo, Isolation, Standard Deviation

Journal: PLoS Pathogens

Article Title: DAMP Molecule S100A9 Acts as a Molecular Pattern to Enhance Inflammation during Influenza A Virus Infection: Role of DDX21-TRIF-TLR4-MyD88 Pathway

doi: 10.1371/journal.ppat.1003848

Figure Lengend Snippet: ( A ) Lung sections were prepared (at 3 d post-infection) from IAV infected (2×10 4 pfu/mouse via intra-tracheal route) mice administered with either control IgG (IgG) or anti-S100A9 blocking (neutralizing) antibody (S100A9 Ab) (24 h prior to IAV inoculation, 2 mg of antibody/mouse administered via i.p route). For each experimental group lung sections were prepared from three control IgG treated mice (+IAV) and three S100A9 Ab treated mice (+IAV). The lung sections were used for TUNEL staining. Image J software was used to calculate TUNEL-positive areas (representing apoptosis) in the lung sections as detailed in the methods section. The data is presented as percent apoptotic area. The percent apoptotic area was calculated from nine areas/lung section as detailed in the methods section. The values were compiled to calculate the percent apoptotic area in IAV infected IgG treated mice vs. IAV infected S100A9 Ab treated mice, * p = 0.0164 by Student's t test. ( B ) A representative TUNEL staining of lung sections from IAV infected mice administered with either IgG or S100A9 Ab. The apoptotic nuclei (representing apoptosis) are indicated with red arrows. ( C ) A schematic model depicting the role of extracellular S100A9 and DDX21/TRIF/S100A9/TLR4/MyD88 signaling network in exaggerating lung disease during IAV infection. PM, plasma membrane; NM, nuclear membrane.

Article Snippet: The plates were washed three times with PBST and incubated with either goat anti-mouse IgG (300 ng/well) (R&D) (for mouse S100A9) or mouse anti-human IgG (50 ng/well) (R&D) (for human S100A9) in PBST+0.1% BSA for 2 h at room temperature.

Techniques: Infection, Blocking Assay, TUNEL Assay, Staining, Software

Journal: The Journal of Clinical Investigation

Article Title: S100A8/A9 regulates CD11b expression and neutrophil recruitment during chronic tuberculosis

doi: 10.1172/JCI130546

Figure Lengend Snippet: B6 mice (n = 3–5) were aerosol infected with approximately 100 CFU Mtb HN878. (A) Lung S100A8/A9 levels were determined by ELISA at dpi. (B) The number of neutrophils was correlated with the levels of S100A8/A9 using a linear regression of the means. (C) Kinetics of S100A8 and S100A9 mRNA expression over time, expressed as log2 fold change (FC) between bin-matched progressors (n = 44) and controls (n = 106) and modeled as nonlinear splines (dotted lines). Light green shading represents 99% CI and dark green shading 95% CI for the temporal trends, computed by performing 2000 spline fitting iterations after bootstrap resampling from the full data set. The deviation time (day), calculated as the time point at which the 99% CI deviates from a log2 fold change of 0, is indicated by the vertical red line. Pulmonary TB patients were enrolled and treated with standard first-line TB regimen. Serum samples were collected at time of diagnosis (0) and at weeks 2, 6, and 26 during treatment. S100A8/A9 levels were determined by ELISA in patients that were (D) successfully treated and cured (n = 34), (E) successfully treated but relapsed (n = 10), and (F) cured (n = 34) versus failed treatment (n = 10) at 2 weeks after treatment initiation. (A) Student’s t test between 0 and indicated dpi; (B) linear regression of the means; (D–F) mixed effects analysis of 1-way ANOVA with Dunnett’s posttest. Figures depict 1 experiment representative of 2 or combined data from multiple experiments. Data points represent the mean ± SEM of values. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Article Snippet: Neutrophils were isolated from the bone marrow of B6 or S100A9 KO using the Mouse Neutrophil Isolation Kit (Miltenyi Biotec).

Techniques: Aerosol, Infection, Enzyme-linked Immunosorbent Assay, Expressing, Biomarker Discovery

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: (A) Single S100A8 or S100A9 yeast transformants or (B) cotransformants with plasmids mCherry S100A8/ GFP S100A9 were grown overnight in SG, and images were obtained with a fluorescence microscope. (C) TCA precipitates of extracts from cells growing on glucose or galactose medium were separated by 10% SDS-PAGE and analyzed by Western blot.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Fluorescence, Microscopy, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: Fluorescent microscope images of GFP S100A8 and GFP S100A9 (green) after (A) 2 days or (B) 4 days of induction. Lipophilic dye FM4-64 was used to visualize vacuoles (red). (C) Quantification of the percent of cells with GFP, GFP S100A8 or GFP S100A9 foci in the vacuole or in the cytoplasm, following 2 and 4 days of induction.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Microscopy

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: GFP (green) and mCherry (red) fluorescent microscope images of mCherry S100A8/ GFP S100A9 cotransformed cells after 2 or 4 days of induction.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Microscopy

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: (A) Confocal images of GFP, GFP S100A8 or GFP S100A9 (green) after 2 days of induction in pep4Δ mutants cells. Lipophilic dye FM4-64 was used to visualize vacuoles (red). (B) Quantification of the percent of cells with GFP, GFP S100A8 or GFP S100A9 foci in the vacuole of pep4 Δ cells, following 2 days of induction.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques:

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: (A) NI, noninduced control (Lanes 1, 4, 7, 10). Extracts of cells induced for 2 or 4 days to produce GFP S100A8 (Lanes 2 and 3), GFP S100A9 (Lanes 5 and 6) and mCherry S100A8/ GFP S100A9 (Lanes 8, 9 and 11,12) were separated on a native gel and analyzed by Western blot. (B) Semi-denaturing agarose detergent gel. After 2 days of induction GFP S100A8 (Lane 1), GFP S100A9 (Lane 3) or cotransformants mCherry S100A8/ GFP S100A9 (Lanes 7, 8 and 10, 11) formed SDS-stable aggregates in yeast cells. Boiling (+) the samples led to full soloubilization of aggregates to the monomeric form (Lanes 2 and 4). Total cell extracts (180 µg) were resolved using SDD-AGE. Blots were probed with anti-GFP or mCherry antibodies. Total cell extract of Q103 GFP cells (90 µg) was prepared after 24 h of induction (lane 5). (C) Filter retardation assay of cells grown for 3 and 5 days under inducing conditions. Loading control was visualized by CBB staining. Empty vector-transfected cells were used as control. (D) Spheroplasts of control and induced cells stained with ThT after 3 days of incubation on galactose plates.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Control, Western Blot, Staining, Plasmid Preparation, Transfection, Incubation

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: (A) Ten-fold dilutions of yeast cells transformed with p GFP-S100A8 , p GFP-S100A9 or both plasmids were plated on glucose (non-inducing) or galactose (inducing) plates and photographed after 72 h. (B) Non-tagged proteins as in A.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Transformation Assay

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: (A) wild type and cdc53-1 ts mutant cells expressing p YES2-S100A8 or p YES2-S100A9 were spotted on glucose or galactose plates and photographed after 72 h. (B ) Semi-denaturing agarose gel. After 2 days of induction GFP S100A9 forms aggregates in wild type and ts strain cdc53-1 at 30°C and 32°C. Total cell extracts (180 µg) were resolved using SDD-AGE. (C ) Ten-fold dilutions of cdc53-1 , cdc34-2 , srp1-31, and sec27-1 yeast cells transformed with p TET-S100A8 or p TET-S100A9 were spotted on SD plates with (inducing) or without (non-inducing) 5 µg/ml doxycycline and photographed after 72 h.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Mutagenesis, Expressing, Agarose Gel Electrophoresis, Transformation Assay

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: (A) Viability of wild type or hsp104Δ yeast in the presence of S100A8, S100A9 and S100A8/9 proteins. p YES2-S100A8 , p YES2-S100A9 or both plasmids were expressed in wild type or hsp104Δ mutant cells. Viability was monitored using the spot test assay on inducing (galactose) or noninducing (glucose) plates. (B) Ten-fold dilutions of wild type cells or hsp104Δ mutants transformed with p GALSc104(WT) and with p YES2-S100A8, p YES2-S100A9 or both S100 plasmids were plated on glucose (non-inducing) or galactose (inducing) plates. (C) Confocal images of GFP S100A8 and GFP S100A9 after 2 days of induction in wild type or Δhsp104 mutant cells. (D) Cell extracts were prepared from wild type or hsp104Δ mutant cells expressing p GFP-S100A8 or p GFP-S100A9 after 2 days of induction. Extracts were incubated in 2% SDS sample buffer with (+) or without (−) boiling, loaded on agarose gels, and analyzed by Western blot using anti-GFP antibodies to detect the S100A8 and S100A9 proteins.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Mutagenesis, Spot Test, Transformation Assay, Expressing, Incubation, Western Blot

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: Cells expressing empty vector (p YES2), p YES2-S100A8 , p YES2-S100A9 , or cotransformants with p YES2-S100A8/ p YES2-S100A9 in sse1, sse2, ssa1, ssa2, ssa3, hsp26, and ydj1 mutants and the isogenic wild type parent were spotted on galactose and glucose plates and photographed after 72 h.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Expressing, Plasmid Preparation

Journal: Journal of Korean medical science

Article Title: S100 Calcium-Binding Protein A9, a Potential Novel Diagnostic Biomarker for Idiopathic Pulmonary Fibrosis.

doi: 10.3346/jkms.2024.39.e13

Figure Lengend Snippet: Fig. 3. S100A9 levels in serum from IPF patients and relationship with survival rates. (A) Serum S100A9 levels were detected in 19 pf 40 HCs and in 53 of 90 IPF patients. Data are expressed as medians with 25.0% and 75.0% quartiles, with statistical significance denoted as *P < 0.05 vs. HCs. (B) Correlation between S100A9 levels in serum and BALF (r = 0.320, P = 0.023). (C) Kaplan–Meier survival curves comparing survival rates between IPF patients with S100A9 levels > 0.077 ng/mL (n = 29, dotted line) and those with S100A9 levels ≤ 0.077 (n = 53, solid line) ng/mL (hazard ratio, 2.52; 95.0% confidence interval, 1.15–5.51; P = 0.013). S100A9 = S100 calcium-binding protein A9, IPF = idiopathic pulmonary fibrosis, HC = healthy control, BALF = bronchoalveolar lavage fluid.

Article Snippet: The sections were then incubated overnight at 4°C with monoclonal mouse anti-human S100A9 antibody (1:200 dilution; Novus Biological, Littleton, CA, USA) and polyclonal mouse anti-human α-SMA antibody (1:200 dilution; Abcam, Cambridge, MA, USA).

Techniques: Binding Assay, Control

Journal: Journal of Korean medical science

Article Title: S100 Calcium-Binding Protein A9, a Potential Novel Diagnostic Biomarker for Idiopathic Pulmonary Fibrosis.

doi: 10.3346/jkms.2024.39.e13

Figure Lengend Snippet: Fig. 4. Immunofluorescence staining of S100A9 in lung tissues and BALF cells of IPF patients. (A) S100A9 (green) and α-SMA (red) were co-stained with PE-(red) or FITC-conjugated antibodies (green), respectively (200× magnification). (B) Images show colocalization of pan-macrophage marker CD163 (Alexa 488) (green) and S100A9 (Alexa 594) (red), along with DAPI-stained nuclei (blue) (400× magnification). Control: lung tissue obtained from normal lungs of the patients who underwent surgery for stage I or II lung cancer. BALF = bronchoalveolar lavage fluid, IPF = idiopathic pulmonary fibrosis, S100A9 = S100 calcium-binding protein A9, SMA = smooth muscle actin, PE = phycoerythrin, FITC = fluorescein isothiocyanate, DAPI = 4′,6-diamidino-2-phenylindole, H&E = hematoxylin & eosin.

Article Snippet: The sections were then incubated overnight at 4°C with monoclonal mouse anti-human S100A9 antibody (1:200 dilution; Novus Biological, Littleton, CA, USA) and polyclonal mouse anti-human α-SMA antibody (1:200 dilution; Abcam, Cambridge, MA, USA).

Techniques: Immunofluorescence, Staining, Marker, Control, Binding Assay

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

Article Title: IL-37 ameliorates the inflammatory process in psoriasis by suppressing proinflammatory cytokine production.

doi: 10.4049/jimmunol.1300047

Figure Lengend Snippet: FIGURE 1. Effect of IL-37 on the production of CXCL8, IL-6, and S100A7 upon stimulation of keratinocytes with M5. The control keratinocytes or kerati- nocytes transfected with pNull or pIL-37 were stimulated with M5. (A) The ex- pression of IL-37 in vitro was detected by Western blot, Mr of IL-37 was ∼34 kDa. Expression of IL-37 in keratino- cyte lysates after overnight recovery from transfection (left panel). Duration of IL-37 expression by pIL-37–trans- fected keratinocytes at 24, 48, and 72 h posttransfection (right panel). Effect of IL-37 on expression of CXCL8 (B), IL-6 (C), and S100A7 (D). Level of CXCL8 (B), IL-6 (C), and S100A7 (D) gene transcripts (left panels). Total RNA was obtained at 0, 6, 12, and 24 h poststim- ulation with M5, and QRT-PCR was carried out for CXCL8, normalized us- ing the housekeeping gene, and ex- pressed as the fold change relative to the control group at 0 h. Level of CXCL8 (B) and IL-6 (C) secreted into the culture supernatants. (D) Level of S100A7 in lysates (right panel). At the indicated time points, levels of CXCL8 and IL-6 were measured by ELISA, and S100A7 was detected by Western blot (Mr of S100A7 was ∼11 kDa). The data are rep- resentative of experiments that were car- ried out in triplicate. *p , 0.05, **p , 0.01, ***p , 0.001, versus control kera- tinocytes at the same time point; #p , 0.05, ##p , 0.01, ###p , 0.001, versus pNull group at the same time point. ns, not significant compared with the con- trol or pNull group.

Article Snippet: The expression of IL-37 and S100A7 in keratinocytes was detected with mouse anti-DDK mAb (Stratagene, Santa Clara, CA) (23) and anti-human S100A7 mAb (Imgenex, San Diego, CA).

Techniques: Control, Transfection, In Vitro, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: S100A6 Overexpression Is Associated with Poor Prognosis and Is Epigenetically Up-Regulated in Gastric Cancer

doi: 10.2353/ajpath.2010.091217

Figure Lengend Snippet: Immunohistochemical staining of S100A6 in primary gastric carcinoma, lymph node metastases, and liver metastatic nodules. A: The left large image is the strong S100A6 expression in tumor (×40), and the right two images are high-power images, showing S100A6 staining in the central part (upper) and invasion front (lower) of primary tumor tissues (×400). B and C: Strong S100A6 staining in lymph node metastases (×100) and liver metastatic nodules (×200).

Article Snippet: Laser Confocal Scanning For double staining, mouse anti-human S100A6 antibody (1:500, Abnova) and rabbit anti-human Ki-67 antibody (1:100, RM-9106-S0, LabVision) were used as primary antibodies.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal:

Article Title: S100A6 Overexpression Is Associated with Poor Prognosis and Is Epigenetically Up-Regulated in Gastric Cancer

doi: 10.2353/ajpath.2010.091217

Figure Lengend Snippet: Association of S100A6 Expression with Clinicopathological Parameters in Gastric Cancer Patients

Article Snippet: Laser Confocal Scanning For double staining, mouse anti-human S100A6 antibody (1:500, Abnova) and rabbit anti-human Ki-67 antibody (1:100, RM-9106-S0, LabVision) were used as primary antibodies.

Techniques: Expressing

Journal:

Article Title: S100A6 Overexpression Is Associated with Poor Prognosis and Is Epigenetically Up-Regulated in Gastric Cancer

doi: 10.2353/ajpath.2010.091217

Figure Lengend Snippet: Kaplan-Meier survival curves for 166 patients with gastric cancer according to S100A6 expression.

Article Snippet: Laser Confocal Scanning For double staining, mouse anti-human S100A6 antibody (1:500, Abnova) and rabbit anti-human Ki-67 antibody (1:100, RM-9106-S0, LabVision) were used as primary antibodies.

Techniques: Expressing

Journal:

Article Title: S100A6 Overexpression Is Associated with Poor Prognosis and Is Epigenetically Up-Regulated in Gastric Cancer

doi: 10.2353/ajpath.2010.091217

Figure Lengend Snippet: Multivariate Analysis of Prognostic Factors by Cox Proportional Hazard Model

Article Snippet: Laser Confocal Scanning For double staining, mouse anti-human S100A6 antibody (1:500, Abnova) and rabbit anti-human Ki-67 antibody (1:100, RM-9106-S0, LabVision) were used as primary antibodies.

Techniques: Expressing

Journal:

Article Title: S100A6 Overexpression Is Associated with Poor Prognosis and Is Epigenetically Up-Regulated in Gastric Cancer

doi: 10.2353/ajpath.2010.091217

Figure Lengend Snippet: Ultrastructural localization of S100A6 expression in gastric cancer tissues and protein expression in human gastric carcinoma (T) and matched nonneoplastic mucosa (N). A: Immunofluorescence staining captured by laser confocal scanning in gastric cancer tissue, including S100A6, Ki-67, DAPI, and merged image. S100A6 subcellular localization includes cytoplasm, whole nucleus, and nucleolus (indicated by white arrowheads). Scale bars, 10 μm. B: Immunoelectron microscopy: cancer cells display strong immunoreactivity as indicated by gold labeling particles throughout the cytoplasm including the endoplasmic reticulum (ER+), and within the nucleus such as nucleoplasm and nucleolus (indicated by black arrowheads). Scale bars, 0.2 μm. C indicates cytoplasm; Nu, nucleus; Nus, nucleolus. C: S100A6 expression in nuclear extracts (n) and cytoplasmic exactions (c) of matched T and N from four gastric cancer patients.

Article Snippet: Laser Confocal Scanning For double staining, mouse anti-human S100A6 antibody (1:500, Abnova) and rabbit anti-human Ki-67 antibody (1:100, RM-9106-S0, LabVision) were used as primary antibodies.

Techniques: Expressing, Immunofluorescence, Staining, Immuno-Electron Microscopy, Labeling

Journal:

Article Title: S100A6 Overexpression Is Associated with Poor Prognosis and Is Epigenetically Up-Regulated in Gastric Cancer

doi: 10.2353/ajpath.2010.091217

Figure Lengend Snippet: The expression of S100A6 in the gastric cancer cell lines. A: Quantitative assessment (Log10 value) of S100A6 mRNA levels by real-time PCR in BGC823, SGC7901, KATO3, AGS, MKN45, RF1, and RF48 cell lines, each sample was run thrice, SD are shown. B: Immunocytochemical staining of S100A6 in gastric cancer cell lines (×400). C: The S100A6 protein expression in gastric cancer cell lines by Western blot.

Article Snippet: Laser Confocal Scanning For double staining, mouse anti-human S100A6 antibody (1:500, Abnova) and rabbit anti-human Ki-67 antibody (1:100, RM-9106-S0, LabVision) were used as primary antibodies.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Staining, Western Blot

Journal:

Article Title: S100A6 Overexpression Is Associated with Poor Prognosis and Is Epigenetically Up-Regulated in Gastric Cancer

doi: 10.2353/ajpath.2010.091217

Figure Lengend Snippet: The CpG sites methylation status in the MKN45, BGC823, SGC7901, KATO3, AGS, RF1, and RF48 cell lines. A: Schematic representation of CpG dinucleotide distribution in 1646bp S100A6 gene and its 1000 bp upstream. B: The methylation status of the CpG sites in the P/E1 and E2/I2 regions. Each bar represents one CpG pair: black fully filled bar, cytosine residue methylated; white bar, cytosine residue unmethylated; half-filled bar: cytosine residue partially methylated. C: The different methylation status of the seventh and eighth CpG sites in the P/E1 region and the second to fifth CpG sites in the E2/I2 region. The red arrows represent the methylation positive (upper) and methylation negative (lower) CpG sites respectively.

Article Snippet: Laser Confocal Scanning For double staining, mouse anti-human S100A6 antibody (1:500, Abnova) and rabbit anti-human Ki-67 antibody (1:100, RM-9106-S0, LabVision) were used as primary antibodies.

Techniques: Methylation, Residue

Journal:

Article Title: S100A6 Overexpression Is Associated with Poor Prognosis and Is Epigenetically Up-Regulated in Gastric Cancer

doi: 10.2353/ajpath.2010.091217

Figure Lengend Snippet: Methylation status of CpG sites in the gastric cancer and nonneoplastic mucosa. A: Percentage of positive methylation in the second to fifth CpG sites of E2/I2 region in paired gastric cancer and nonneoplastic mucosa. B: Average S100A6 expression value of 46 gastric cancer tissues and 20 nonneoplastic mucosa. Error bars indicate SD. C: The expression difference of S100A6 between group one and group two. Group one: cancer tissues with the second to fifth sites of the E2/I2 region entirely methylated; group two: cancer tissues with at least a single CpG site of this region unmethylated. Error bars indicate SD.

Article Snippet: Laser Confocal Scanning For double staining, mouse anti-human S100A6 antibody (1:500, Abnova) and rabbit anti-human Ki-67 antibody (1:100, RM-9106-S0, LabVision) were used as primary antibodies.

Techniques: Methylation, Expressing

Journal:

Article Title: S100A6 Overexpression Is Associated with Poor Prognosis and Is Epigenetically Up-Regulated in Gastric Cancer

doi: 10.2353/ajpath.2010.091217

Figure Lengend Snippet: The influence of the 5-azacytidine or TSA on the S100A6 expression in MKN45, RF1, and RF48 cells. A: The relative quantification (Log10 value) of S100A6 mRNA increases in the 5, 10 μmol/L 5-azacytidine-treated RF1, RF48, and MKN45 cells. B: PCR amplification of the ChIP products from TSA-treated RF1, RF48, and MKN45 cells, after precipitating the sonicated lysate with antibodies against modified histone H3: Ac lys9, anti-acetyl-H3 (lysine 9); and Ac H3, anti-acetyl-H3. C: The relative quantification of S100A6 mRNA change in the TSA-treated RF1, RF48, and MKN45 cells. Statistical analysis was performed using the Student’s t-test. Data shown are the mean ± SD of at least three independent experiments. **P < 0.01.

Article Snippet: Laser Confocal Scanning For double staining, mouse anti-human S100A6 antibody (1:500, Abnova) and rabbit anti-human Ki-67 antibody (1:100, RM-9106-S0, LabVision) were used as primary antibodies.

Techniques: Expressing, Quantitative Proteomics, Amplification, Sonication, Modification