Journal: OncoTargets and Therapy

Article Title:

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling

doi: 10.2147/ott.s192250

Figure Lengend Snippet: Figure 1 S100A9 promotes prostate cancer cell invasion and β1 integrin expression through interaction with TLR4. PC-3 and DU-145 cells were treated with S100A9 (20 µg/ml) for 48 h. (A) PC-3 and DU-145 cells invasion was measured by transwell invasion assay. (B) The mRNA and protein levels of β1 integrin were determined by qPCR or Western blot. PC-3 and DU-145 cells were treated with S100A9 (20 µg/ml) for 48 h. (C, D) Cell extracts were immunoprecipitated (IP) with control mouse IgG, mouse anti-S100A9 antibody. Immunoblot (IB) was used to detect S100A9, TLR4 and RAGE. PC-3 and DU-145 cells were transfected with TLR4 or control siRNA. (E) TLR4 expression was examined by Western blot after 48 h siRNA transfection. PC-3 and DU-145 cells were transfected with TLR4 or control siRNA followed by stimulation with S100A9. (F) Tumor cell invasion was measured by transwell invasion assay. (G) The mRNA and protein levels of β1 integrin were determined by qPCR or Western blot. Scale bar 50 μ. Magnifcation×200. Data are represented as the mean ± S.E.M. *p<0.05.

Article Snippet: Antibodies and reagents The recombinant human S100A9 (9254-S9), human S100A9 ELISA Kit (DY5578) were purchased from R&D Systems (R&D Systems, Shanghai, China).

Techniques: Expressing, Transwell Invasion Assay, Western Blot, Immunoprecipitation, Control, Transfection

Journal: OncoTargets and Therapy

Article Title:

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling

doi: 10.2147/ott.s192250

Figure Lengend Snippet: Figure 2 NF-κB mediates S100A9-induced prostate cancer cell β1 integrin up-regulation. PC-3 and DU-145 cells transfected with or without TLR4 siRNA or control siRNA, were transfected with NF-κB-luciferase reporter plasmid, and treated with S100A9 (20 µg/ml) for 48 h. (A, B) Activity of NF-κB was detected by measuring the relative activity of luciferase. PC-3 and DU-145 cells were treated with or without BAY11-7082 (5µM) for 30 min. Then cells were treated with or without S100A9 (20 µg/ ml) for 48 h. (C) The mRNA and protein levels of β1 integrin were determined by qPCR or Western blot. Data are represented as the mean ± S.E.M. *p<0.05.

Article Snippet: Antibodies and reagents The recombinant human S100A9 (9254-S9), human S100A9 ELISA Kit (DY5578) were purchased from R&D Systems (R&D Systems, Shanghai, China).

Techniques: Transfection, Control, Luciferase, Plasmid Preparation, Activity Assay, Western Blot

Journal: OncoTargets and Therapy

Article Title:

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling

doi: 10.2147/ott.s192250

Figure Lengend Snippet: Figure 3 S100A9 promotes prostate cancer cell invasion via integrin β1/FAK signaling. PC-3 and DU-145 cells were treated with or without BAY11-7082 (5µM) for 30 min. Then cells were treated with or without S100A9 (20 µg/ml) for 48 h. (A) Fibronectin expression was determined by Western blot. PC-3 and DU-145 cells were treated with or without S100A9 (20 µg/ml) for 48 h. (B) Supernatant fibronectin (FN) concentration was determined by ELISA. PC-3 and DU-145 cells were treated with or without S100A9 (20 µg/ml) for 30 min. (C) The phosphorylation of FAK was measured by Western blot. PC-3 and DU-145 cells were transfected with control siRNA or integrin β1-specific siRNA for 48 h. Then cells were treated with or without S100A9 (20 µg/ml) for 30 min, the expression of integrin β1 (D) or phosphorylation of FAK (E) was measured by Western blot. PC-3 and DU-145 cells were transfected with control siRNA or integrin β1-specific siRNA for 48 h. Then cells were treated with or without S100A9 (20 µg/ml) for 48 h. (F) The invasion activity were measured by transwell invasion assay. (G) PC-3 and DU- 145 cells were treated with β1 integrin functional blocking antibody MAB13 (50 μg/mL) or control IgG (50 μg/mL) for 30 min and then treated with or without S100A9 (20 µg/ml) for 30 min and the phosphorylation of FAK was measured by Western blot. (H) PC-3 and DU-145 cells were treated with MAB13 (50 μg/mL) or control IgG (50 μg/mL) for 30 min and then treated with or without S100A9 (20 µg/ml) for 48 h for invasion. (I) PC-3 and DU-145 cells were pretreated for 30 min with FAK inhibitor, PF562271 (100 nM) followed by stimulation with S100A9 (20 µg/ml) for 48 h for invasion. Data are represented as the mean ± S.E.M. *p<0.05.

Article Snippet: Antibodies and reagents The recombinant human S100A9 (9254-S9), human S100A9 ELISA Kit (DY5578) were purchased from R&D Systems (R&D Systems, Shanghai, China).

Techniques: Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Transfection, Control, Activity Assay, Transwell Invasion Assay, Functional Assay, Blocking Assay

Journal: OncoTargets and Therapy

Article Title:

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling

doi: 10.2147/ott.s192250

Figure Lengend Snippet: Figure 4 S100A9 induces prostate cancer cell metastasis in vivo. DU-145 cells were transfected with pcDNA3.1 or pc DNA-S100A9 plasmid. (A, B) The expression or secretion of S100A9 was determined by Western blot and ELISA. The cells were injected to nude mice via tail vein. 30 days after inoculation, nude mice were sacrificed. (C) Expressions of S100A9 and integrin β1 in xenograft tumors were detected by immunohistochemistry. (D) Expressions of FAK, p-FAK, NF-kB p65 and p-NF-kB p65 in xenograft tumors were determined by Western blot. (E) Micrometastatic tumors in the lungs of mouse xenografts were counted and subjected to H and E staining. Scale bar 50 μ. Magnifcation×200. Data are represented as the mean ± S.E.M.*p<0.05.

Article Snippet: Antibodies and reagents The recombinant human S100A9 (9254-S9), human S100A9 ELISA Kit (DY5578) were purchased from R&D Systems (R&D Systems, Shanghai, China).

Techniques: In Vivo, Transfection, Plasmid Preparation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Immunohistochemistry, Staining

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: 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