Journal: Infection and Immunity

Article Title: Epithelial Cell-Derived S100 Calcium-Binding Proteins as Key Mediators in the Hallmark Acute Neutrophil Response during Candida Vaginitis ▿

doi: 10.1128/IAI.00388-10

Figure Lengend Snippet: Protein integrity scores from excised bands at 14 and 6 kDa a

Article Snippet: Recombinant mouse S100A8 and S100A9 (10 ng; R&D Systems) were included as references for the molecular weight and antibody specificity. (iv) ELISA.

Techniques: Derivative Assay

Journal: Infection and Immunity

Article Title: Epithelial Cell-Derived S100 Calcium-Binding Proteins as Key Mediators in the Hallmark Acute Neutrophil Response during Candida Vaginitis ▿

doi: 10.1128/IAI.00388-10

Figure Lengend Snippet: S100A8 and S100A9 are present in vivo postinoculation. (A) Western blot. Proteins in lavage fluids from estrogenized inoculated mice with high PMN levels (H) or low PMN levels (L) or estrogenized uninoculated mice (U) were separated and visualized by Western blotting with anti-S100A8 (left panel) or anti-S100A9 (right panel) antibodies. Recombinant mouse S100A8 (rA8) and S100A9 (rA9) were included as positive controls. MW denotes molecular mass markers. Masses are indicated in kilodaltons on the right side of the MW lanes. The figure shows a representative image of three repeat experiments testing lavage samples collected on day 4 postinoculation. (B and C) ELISA. Diluted lavage fluid from estrogenized inoculated mice with high PMN levels or low PMN levels or uninoculated mice were evaluated for S100A8 and S100A9 concentrations by ELISA. The results are cumulative data of three repeat experiments testing lavage samples collected on day 4 postinoculation. **, P < 0.005. ***, P < 0.001. LF, lavage fluid. SEM, standard error of the mean.

Article Snippet: Recombinant mouse S100A8 and S100A9 (10 ng; R&D Systems) were included as references for the molecular weight and antibody specificity. (iv) ELISA.

Techniques: In Vivo, Western Blot, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Infection and Immunity

Article Title: Epithelial Cell-Derived S100 Calcium-Binding Proteins as Key Mediators in the Hallmark Acute Neutrophil Response during Candida Vaginitis ▿

doi: 10.1128/IAI.00388-10

Figure Lengend Snippet: Presence of S100A8 and S100A9 on vaginal epithelial cells after interaction with Candida. (A and B) Vaginal tissue sections (A) and cytospin preparations of cellular fractions in pooled lavage fluid from estrogenized uninoculated or inoculated mice with high PMN levels or low PMN levels (B) were stained with anti-S100A8, anti-S100A9, anti-AE1/AE3 (pan-epithelial cell marker), or isotype control antibodies. Images are shown at ×400 magnification. Arrows represent epithelial cells positively stained for S100A8 or S100A9. Images show a representative result of three repeat experiments testing specimens collected on day 7 postinoculation. (C and D) The numbers of positively stained epithelial cells for S100A8 and S100A9 were counted in five nonadjacent fields per slide at ×100 magnification and averaged. The results are cumulative data of three experiments. *, P < 0.05; **, P < 0.01. SEM, standard error of the mean.

Article Snippet: Recombinant mouse S100A8 and S100A9 (10 ng; R&D Systems) were included as references for the molecular weight and antibody specificity. (iv) ELISA.

Techniques: Staining, Marker, Control

Journal: Infection and Immunity

Article Title: Epithelial Cell-Derived S100 Calcium-Binding Proteins as Key Mediators in the Hallmark Acute Neutrophil Response during Candida Vaginitis ▿

doi: 10.1128/IAI.00388-10

Figure Lengend Snippet: Vaginal epithelial cells are a source of S100A8 and S100A9 during infection. (A and B) S100A8 and S100A9 mRNA expression by vaginal epithelial cells. Total RNA was extracted from vaginal epithelial cells from estrogenized uninoculated or inoculated mice with high PMN levels or low PMN levels and reverse transcribed into cDNA. S100A8 and S100A9 mRNA expression was quantified and normalized to β-actin mRNA expression. The results are expressed as the fold increase over expression in cells from estrogenized uninoculated mice. The results are cumulative data of 3 experiments testing vaginal epithelial cells collected on day 4 postinoculation. *, P = 0.0024. SEM, standard error of the mean. (C) S100A8 and S100A9 protein production by vaginal epithelial cells. Proteins in vaginal epithelial cell lysates from estrogenized inoculated mice with high PMN levels (H) or low PMN levels (L) or estrogenized uninoculated mice (U) were separated and visualized by Western blotting with anti-S100A8 (left panel), anti-S100A9 (right panel) or β-actin (bottom panel) antibodies. Recombinant mouse S100A8 (rA8) and S100A9 (rA9) were included as positive controls. MW denotes molecular mass markers. Masses are indicated in kilodaltons on the right side of the MW lanes. Images show a representative result of three repeat experiments testing vaginal epithelial cells collected on day 4 postinoculation.

Article Snippet: Recombinant mouse S100A8 and S100A9 (10 ng; R&D Systems) were included as references for the molecular weight and antibody specificity. (iv) ELISA.

Techniques: Infection, Expressing, Reverse Transcription, Over Expression, Western Blot, Recombinant

Journal: Infection and Immunity

Article Title: Epithelial Cell-Derived S100 Calcium-Binding Proteins as Key Mediators in the Hallmark Acute Neutrophil Response during Candida Vaginitis ▿

doi: 10.1128/IAI.00388-10

Figure Lengend Snippet: Role of mouse S100A8 and S100A9 in PMN chemotaxis. Pooled lavage fluid with positive chemotactic activity was incubated with anti-S100A8 or anti-S100A9 antibodies and tested in the PMN chemotaxis assay. The numbers of PMNs migrating to the bottom chamber were quantified by microscopic counts. The results are expressed as the percentage of control compared to the PMN migration by the same fluid incubated with the isotype control IgG antibody. Figure shows cumulative data from three repeats. *, P < 0.05. SEM, standard error of the mean.

Article Snippet: Recombinant mouse S100A8 and S100A9 (10 ng; R&D Systems) were included as references for the molecular weight and antibody specificity. (iv) ELISA.

Techniques: Chemotaxis Assay, Activity Assay, Incubation, Control, Migration

Journal: International journal of molecular sciences

Article Title: Inflammation of Dry Eye Syndrome: A Cellular Study of the Epithelial and Macrophagic Involvement of NFAT5 and RAGE.

doi: 10.3390/ijms241311052

Figure Lengend Snippet: Figure 2. Induction of S100 proteins by hyperosmolarity in different ocular surface epithelial cells. Cells were exposed to a hyperosmolar medium (70 mM NaCl [orange] or 90 mM NaCl [green]) for 24 h and (A) analyzed for mRNA expressions of S100A4, S100A8, and S100A9 via RT-qPCR. (B) After 48 h of hyperosmolarity exposition, the protein releases of S100A4, S100A8, S100A9 and S100A8/A9 were analyzed via ELISA in a Wong–Kilbourne derivative of the Chang conjunctival (WKD) cell line. (C) The mRNA expression of alarmins were analyzed (D) and protein release in a human corneal epithelial (HCE) cell line. The experiment was repeated four times (n = 4). Results are expressed as a fold change between treated and non-treated (NT) conditions and were analyzed via ANOVA statistical test (Kruskal–Wallis), followed by Dunn’s post hoc test: * p = 0.05, ** p = 0.01 and *** p = 0.001. The horizontal line corresponds to the value of the non-treated cells reported as 1 (n = 4).

Article Snippet: Human S100A4 (4137-S4), S100A8 (9876-S8), S100A9 (9254-S9), and MCP1 (279-MC-010/CF) recombinant proteins were obtained from R&D systems (Noyal-Châtillon-sur-Seiche, France).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing

Journal: International journal of molecular sciences

Article Title: Inflammation of Dry Eye Syndrome: A Cellular Study of the Epithelial and Macrophagic Involvement of NFAT5 and RAGE.

doi: 10.3390/ijms241311052

Figure Lengend Snippet: Figure 9. Action of DAMPs on the epithelial cell release of cytokines. Epithelial cells were exposed to a normal medium with protein recombinant (S100A4 [pink], S100A8 [purple], or S1009 [blue] at 0.1 and 1 µg/mL or HMGB1 [green] at 100 or 300 ng/mL) for 48 h and then analyzed for protein release of IL6, IL8, TNFα, and MCP1 via ELLA and Multiplex in (A) a Wong–Kilbourne derivative of the Chang conjunctival cell (WKD) line and (B) a human corneal epithelial (HCE) cell line. The experiment was repeated four times (n = 4). Results are expressed as a fold change between treated and non-treated (NT) conditions and were analyzed via ANOVA statistical test (Kruskal–Wallis), followed by Dunn’s post hoc test. The horizontal line corresponds to the value of the non-treated cells reported as 1.

Article Snippet: Human S100A4 (4137-S4), S100A8 (9876-S8), S100A9 (9254-S9), and MCP1 (279-MC-010/CF) recombinant proteins were obtained from R&D systems (Noyal-Châtillon-sur-Seiche, France).

Techniques: Recombinant, Multiplex Assay

Journal: International journal of molecular sciences

Article Title: Inflammation of Dry Eye Syndrome: A Cellular Study of the Epithelial and Macrophagic Involvement of NFAT5 and RAGE.

doi: 10.3390/ijms241311052

Figure Lengend Snippet: Figure 10. Effects of DAMPs and MCP1 in macrophage migration. In the CytoSlectTM 24-well cell migration assay, THP1 was differentiated for 48 h. (A) Next, macrophages were exposed to S100A4 (2 ng/mL [pink]), S100A8 (3 ng/mL [purple]), S100A9 (2 ng/mL [blue]), HMGB1 (100 ng/mL [green]), or MCP1 (100 ng/mL [yellow]). (B) Macrophages were exposed to the same DAMPs with or without RAGE inhibitors (RAP [angled hatches]) and TLR4 (TAK [horizontal hatches]). The experiment was repeated three times (n = 3). The quantification of cells in the top and bottom of the insert was measured via fluorescence. Results are expressed as a fold change between treated and non-treated (NT) conditions and were analyzed via ANOVA statistical test (Kruskal–Wallis), followed by Dunn’s post hoc test. * p = 0.05. The horizontal line corresponds to the value of the non-treated cells reported as 1.

Article Snippet: Human S100A4 (4137-S4), S100A8 (9876-S8), S100A9 (9254-S9), and MCP1 (279-MC-010/CF) recombinant proteins were obtained from R&D systems (Noyal-Châtillon-sur-Seiche, France).

Techniques: Migration, Cell Migration Assay

Journal: Journal of Biological Chemistry

Article Title: Matrix Metalloproteinase Mmp-1a Is Dispensable for Normal Growth and Fertility in Mice and Promotes Lung Cancer Progression by Modulating Inflammatory Responses

doi: 10.1074/jbc.m112.439893

Figure Lengend Snippet: FIGURE 4. A, difference gel electrophoresis analysis of lung tissue from urethane-treated mice. Overlaying green and red image highlights differences between wild-type and Mmp1a/ mice. Yellow indicates no change, red spots indicate more abundance in knock-out mice, and green spots more abundance in wild-type mice. Selected proteins are labeled with white circles, and bidimensional validation analysis is shown in supplemental Fig. 1. All differential analyzed spotsarelistedinsupplementalTable1.B,Westernblotanalysisextendedtootherurethane-treatedlittermatesshowingtheincreasedCHI3L3andRAGElevels in wild-type and knock-out mice, respectively, as well as the differential processing of CHI3L3 in wild-type lungs no present in the mutant lungs. Load control is shown at the bottom of each panel. C, Western blot analysis of the RAGE ligand S100A8 showing accumulation of different isoforms of this chemokine in lung from knock-out mice. D, in vitro cleavage assays with human MMP-1. Purified S100A8, S100A9, and CHI3L3 (1 g) were incubated with 100 ng of activated MMP-1, which resulted in specific cleavage of S100A8 (*), but not S100A9 and CHI3L3. Recombinant MMP-2 was used as a positive control of S100A8 cleavage.

Article Snippet: Enzymatic Assays—For in vitro proteolysis assays, we used recombinant S100A8 and S100A9 kindly provided by Dr. Philippe Tessier and recombinant CHI3L3 and MMP-1 from R&D Systems.

Techniques: Nucleic Acid Electrophoresis, Knock-Out, Labeling, Biomarker Discovery, Mutagenesis, Control, Western Blot, In Vitro, Purification, Incubation, Recombinant, Positive Control

Journal: Molecular biomedicine

Article Title: Enteric glial cells aggravate the intestinal epithelial barrier damage by secreting S100β under high-altitude conditions.

doi: 10.1186/s43556-023-00143-1

Figure Lengend Snippet: Fig. 1 The levels of EGC biomarkers are significantly increased by HA conditions. a Serum concentrations of GFAP, S100β, GDNF, and NGF in control and HA group participants. b Relative mRNA expression per β-Actin of GFAP, S100β, GDNF, and NGF-β in the small intestines of control and HA group mice. c Immunohistochemical staining of the small intestine for GFAP and S100β. (n = 6–8 mice/group), Mean ± SD represent the findings. ****p < 0.0001, **p < 0.01, *p < 0.05. MOD—Mean Optical Density, IHC - immunohistochemistry

Article Snippet: To explore the role of S100β, MODE-K cells were treated with 5 μM recombinant mouse S100β (Solarbio, P00208) and subjected to hypoxia for 12 h, and control MODE-K cells were subjected to hypoxia alone.

Techniques: Control, Expressing, Immunohistochemical staining, Staining, Immunohistochemistry

Journal: Molecular biomedicine

Article Title: Enteric glial cells aggravate the intestinal epithelial barrier damage by secreting S100β under high-altitude conditions.

doi: 10.1186/s43556-023-00143-1

Figure Lengend Snippet: Fig. 5 S100β mediates the effect of EGCs to aggravate epithelial cell injury under hypoxic conditions. a Effect of exogenous S100β on MODE-K epithelial cell proliferation under hypoxic conditions. b Representative flow cytometric plots of MODE-K cell apoptosis. Annexin V and PI were used to label the cells before they were analyzed by flow cytometry. The figures show what proportion of the total occur in each quadrant. c Statistical analysis of the frequency percentage in late-phase apoptosis. d Relative mRNA expression per β-Actin of IL-6, -10, -1α, -1β, and TNF-α, claudin-1, occludin, and ZO-1. e Occludin and ZO-1 protein expression. Representation of findings and significance as previously stated

Article Snippet: To explore the role of S100β, MODE-K cells were treated with 5 μM recombinant mouse S100β (Solarbio, P00208) and subjected to hypoxia for 12 h, and control MODE-K cells were subjected to hypoxia alone.

Techniques: Flow Cytometry, Expressing

Journal: Journal of Translational Medicine

Article Title: Combining patient proteomics and in vitro cardiomyocyte phenotype testing to identify potential mediators of heart failure with preserved ejection fraction

doi: 10.1186/s12967-016-0774-3

Figure Lengend Snippet: Proteins preferential to either HFpEF or control groups

Article Snippet: Recombinant S100A8 was purchased from Creative BioMart (Shirley, NY).

Techniques: Control, Sequencing, Ubiquitin Proteomics

Journal: Journal of Translational Medicine

Article Title: Combining patient proteomics and in vitro cardiomyocyte phenotype testing to identify potential mediators of heart failure with preserved ejection fraction

doi: 10.1186/s12967-016-0774-3

Figure Lengend Snippet: Representative MS/MS scan for S100A8 peptide sequence ALNSIIDVYHK. Raw m/z spectral images with peak assignments and b and y ion lists along with a representation of peptide sequencing by tandem mass spectrometry

Article Snippet: Recombinant S100A8 was purchased from Creative BioMart (Shirley, NY).

Techniques: Tandem Mass Spectroscopy, Sequencing, Mass Spectrometry

Journal: Journal of Translational Medicine

Article Title: Combining patient proteomics and in vitro cardiomyocyte phenotype testing to identify potential mediators of heart failure with preserved ejection fraction

doi: 10.1186/s12967-016-0774-3

Figure Lengend Snippet: Plasma levels of S100A8 in control vs. HFpEF groups. a S100A8 is found in increased levels in the plasma of subjects with HFpEF vs. control subjects as detected by ELISA. The MCW columns include the control (n = 7) and HFpEF (n = 9) from the discovery cohort and the NWU colums include the control (n = 18) and HFpEF (n = 25) samples from the validation cohort. *p < 0.006 vs MCW Control. # p < 0. 002 vs NWU Control

Article Snippet: Recombinant S100A8 was purchased from Creative BioMart (Shirley, NY).

Techniques: Clinical Proteomics, Control, Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: Journal of Translational Medicine

Article Title: Combining patient proteomics and in vitro cardiomyocyte phenotype testing to identify potential mediators of heart failure with preserved ejection fraction

doi: 10.1186/s12967-016-0774-3

Figure Lengend Snippet: Overview of primary and secondary screening methods to identify potential mediators of HFpEF. a Platelet proteomes were subject to mass spectral analysis and novel proteins were identified. b Human cardiomyocytes derived from induced pluripotent stem cells were used to determine whether proteins that were identified in a had direct effects on cardiomyocytes function in vitro. Purified recombinant protein S100A8 was tested in this assay

Article Snippet: Recombinant S100A8 was purchased from Creative BioMart (Shirley, NY).

Techniques: Derivative Assay, In Vitro, Purification, Recombinant

Journal: Journal of Translational Medicine

Article Title: Combining patient proteomics and in vitro cardiomyocyte phenotype testing to identify potential mediators of heart failure with preserved ejection fraction

doi: 10.1186/s12967-016-0774-3

Figure Lengend Snippet: S100A8-mediated effects on human iPSC-derived cardiomyocytes. a Shows example action potentials recorded from rS100A8 treated iPSC derived human cardiomyocytes. The addition of rS100A8 to the buffer extended the period between action potentials. This period is phase 4; the diastolic membrane potential between action potentials. b rS100A8 exacerbates the arrhythmic tendencies of human cardiomyocytes. c Spontaneous Ca 2+ transients recorded from human cardiomyocytes treated with rS100A8 as indicated by the blue line. rS100A8 significantly delayed the recovery of depolarization. Wash out of rS100A8 reversed these effects

Article Snippet: Recombinant S100A8 was purchased from Creative BioMart (Shirley, NY).

Techniques: Derivative Assay, Membrane