Journal: PLoS ONE

Article Title: The Acute Neutrophil Response Mediated by S100 Alarmins during Vaginal Candida Infections Is Independent of the Th17-Pathway

doi: 10.1371/journal.pone.0046311

Figure Lengend Snippet: Vaginal lavage fluid from uninoculated or inoculated wild-type, IL-23p19 −/− , IL-17RA −/− and IL-22 −/− mice with high PMNs were evaluated for (A) S100A8 and (B) S100A9 concentrations by ELISA. The results are cumulative data of 1 to 3 repeat experiment(s) testing lavage samples collected on day 7 post-inoculation. LF, lavage fluid. SEM, standard error of the mean.

Article Snippet: Briefly, tissues were treated with peroxidase, goat serum, avidin and biotin blocking buffers and then incubated with monoclonal rat anti-mouse S100A8 or S100A9 antibody (10 μg/ml; R&D Systems), monoclonal mouse anti-human AE1/AE3 antibody (epithelial cytokeratin markers, 5 μg/ml; MP Biomedicals, Solon, OH), or isotype controls (rat IgG2a, rat IgG2b and mouse IgG1) overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: The Acute Neutrophil Response Mediated by S100 Alarmins during Vaginal Candida Infections Is Independent of the Th17-Pathway

doi: 10.1371/journal.pone.0046311

Figure Lengend Snippet: Vaginal tissue sections from uninoculated or inoculated mice with high PMNs were stained with anti-S100A8 or S100A9 antibodies. Images are shown at ×400 magnification. Arrows represent epithelium positively stained for S100A8 or S100A9. Images show representative results of 15 uninoculated and 30 inoculated animals on day 7 post-inoculation.

Article Snippet: Briefly, tissues were treated with peroxidase, goat serum, avidin and biotin blocking buffers and then incubated with monoclonal rat anti-mouse S100A8 or S100A9 antibody (10 μg/ml; R&D Systems), monoclonal mouse anti-human AE1/AE3 antibody (epithelial cytokeratin markers, 5 μg/ml; MP Biomedicals, Solon, OH), or isotype controls (rat IgG2a, rat IgG2b and mouse IgG1) overnight at 4°C.

Techniques: Staining

Journal: Mucosal immunology

Article Title: β-Glucans in food modify colonic microflora by inducing antimicrobial protein, calprotectin, in a Dectin-1-induced-IL-17F-dependent manner.

doi: 10.1038/mi.2017.86

Figure Lengend Snippet: Figure 3 Interleukin-17F (IL-17F) production is decreased in colonic lamina propria (cLP) of Clec7a / mice. (a) Cytokine gene expression in cLP was examined by quantitative real-time reverse transcription PCR (qRT-PCR), and expression levels in Clec7a / mice are indicated relative to those in wild-type (WT) mice (n ¼ 3–8/group). (b,c) IL-17F þCD45 þ cell population in cLP layer from WT or Clec7a / mice was examined by flow cytometry. (n ¼ 4–5/group). (d) S100A8 þ and IL-17F þ cells in whole colonic cells from WT mice were examined by flow cytometry. Data are representatives of two independent experiments, and in a and c are expressed as mean±s.d. *Po0.05 vs. WT control.

Article Snippet: Antibodies against mouse CD11b (M1/70), CD11c (HL3), CD45 (30-F11), Ly6G (1A8), and Ly6C (HK1.4) were obtained from BioLegend (San Diego, CA), antibodies against IL-17F (18F10) and EpCAM-1 (G8.8) were obtained from eBioscience (San Diego, CA), and antibody against S100A8 (cat. no. BAF3059) was obtained from R&D Systems.

Techniques: Gene Expression, Reverse Transcription, Quantitative RT-PCR, Expressing, Flow Cytometry, Control

Journal: Mucosal immunology

Article Title: β-Glucans in food modify colonic microflora by inducing antimicrobial protein, calprotectin, in a Dectin-1-induced-IL-17F-dependent manner.

doi: 10.1038/mi.2017.86

Figure Lengend Snippet: Figure 6 Dectin-1 ligands are not detected in intestinal commensal microbiota. (a) Wild-type (WT) mouse fecal microbiota or Candida albicans (SC5314) were pretreated with non-immune goat or rabbit serum to block nonspecific binding. Then, they were treated with PE-labeled b-glucan- recognizing protein (Pi-BGRP) or the mutant form of the b-glucan recognition site of Pi-BGRP (Pi-BGRP.M), and PE þ population was determined by flow cytometry. (b) Thioglycollate-induced peritoneal macrophages (Mfs) from WT, Clec7a / , Myd88 / , or Clec7a / Myd88 / mice were cocultured with live Alcaligenes faecalis (multiplicity of infection (MOI): 1 or 10) for 24 h and the tumor necrosis factor-a (TNF-a) concentration in the culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA; n ¼ 3/group). (c) S100A8 expression in colonic epithelial cells (cECs) from A. faecalis monocolonized (A. faecalis), germ-free (GF), or specific-pathogen-free SPF mice was examined by flow cytometry (n ¼ 3–4/group). Data in b and c are representatives of two independent experiments and are expressed as mean±s.d.

Article Snippet: Antibodies against mouse CD11b (M1/70), CD11c (HL3), CD45 (30-F11), Ly6G (1A8), and Ly6C (HK1.4) were obtained from BioLegend (San Diego, CA), antibodies against IL-17F (18F10) and EpCAM-1 (G8.8) were obtained from eBioscience (San Diego, CA), and antibody against S100A8 (cat. no. BAF3059) was obtained from R&D Systems.

Techniques: Blocking Assay, Binding Assay, Labeling, Mutagenesis, Flow Cytometry, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: E-Selectin-Dependent Inflammation and Lipolysis in Adipose Tissue Exacerbate Steatosis-to-NASH Progression via S100A8/9

doi: 10.1016/j.jcmgh.2021.08.002

Figure Lengend Snippet: Antibodies

Article Snippet: S100A8/A9 (mouse) , 1:500 (staining) 1:5000 (WB) , Novus Biologicals.

Techniques: Staining

Journal: BMC Immunology

Article Title: CD11b + Ly6C ++ Ly6G - cells show distinct function in mice with chronic inflammation or tumor burden

doi: 10.1186/1471-2172-13-69

Figure Lengend Snippet: Expression of S100A9 in the splenic CD11b + cells. A. Dot-plots from FACS analysis to illustrate the gating to obtain the different CD11b + subpopulations. B. RT-PCR analysis of S100A9 RNA expression in FACS sorted cells from spleen, as indicated. C. Western blot analysis of S100A9 expression on FACS sorted cell populations from spleen. D. Cytospin of splenic CD11b sorted cells were stained with anti-Ly6G in red, anti-Ly6C in purple, anti-S100A9 in green and Hoecst in blue.

Article Snippet: Thereafter, the membranes were incubated with Rat anti-Mouse S100A9 and Rat anti-Actin (RnD Systems) as primary antibody and Rabbit anti-Rat –HRP (SouthernBiotec Birmingham, Alabama, USA) as secondary antibodies and filters developed using ECL kit (GE Healthcare, UK).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, RNA Expression, Western Blot, Staining

Journal: BMC Immunology

Article Title: CD11b + Ly6C ++ Ly6G - cells show distinct function in mice with chronic inflammation or tumor burden

doi: 10.1186/1471-2172-13-69

Figure Lengend Snippet: Tumor microenvironment induces a shift in CD11b + cell populations. A . Proportion of splenic CD11b + cell population in C57BL/6 control animals and animals inoculated 14 days earlier with EL-4 lymphoma cells. B . Proportion of different CD11b + cell populations in spleen and tumor from EL-4 inoculated C57BL/6 mice. C . RT-PCR analysis of S100A9 RNA expression in sorted cells from spleen and EL-4 tumors, as indicated. The threshold cycle number was determined and relative expression compared to the reference gene (β-actin) and the target gene, respectively. D . Western blot analysis of S100A9 expression on sorted cell populations from spleen and EL-4 tumors, as indicated. The Gray Value (Median) from Adobe Photoshop were used to determined the level of S100A9 and the reference gene (β-actin) expression. Statistical analysis was performed using Student’s t -test (*p < 0.05; **p < 0.01;***p < 0.001).

Article Snippet: Thereafter, the membranes were incubated with Rat anti-Mouse S100A9 and Rat anti-Actin (RnD Systems) as primary antibody and Rabbit anti-Rat –HRP (SouthernBiotec Birmingham, Alabama, USA) as secondary antibodies and filters developed using ECL kit (GE Healthcare, UK).

Techniques: Control, Reverse Transcription Polymerase Chain Reaction, RNA Expression, Expressing, Western Blot

Journal: BMC Immunology

Article Title: CD11b + Ly6C ++ Ly6G - cells show distinct function in mice with chronic inflammation or tumor burden

doi: 10.1186/1471-2172-13-69

Figure Lengend Snippet: Chronic inflammatory microenvironment induces a shift of CD11b + cell populations. A . Proportion of splenic CD11b + cell population in BALB/c control animals and animals inoculated 14 days earlier with pristane i.p. B . Proportion of different CD11b + cell populations in spleen and granuloma inoculated 14 days earlier with pristane i.p. C . RT-PCR analysis of S100A9 RNA expression in sorted cells from spleen and granulomas from animals inoculated 14 days earlier with pristane i.p., as indicated. The threshold cycle number was determined and relative expression compared to the reference gene (β-actin) and the target gene, respectively. Statistical analysis was performed using Student’s t -test (*p < 0.05; **p < 0.01;***p < 0.001).

Article Snippet: Thereafter, the membranes were incubated with Rat anti-Mouse S100A9 and Rat anti-Actin (RnD Systems) as primary antibody and Rabbit anti-Rat –HRP (SouthernBiotec Birmingham, Alabama, USA) as secondary antibodies and filters developed using ECL kit (GE Healthcare, UK).

Techniques: Control, Reverse Transcription Polymerase Chain Reaction, RNA Expression, Expressing

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