Journal: PeerJ

Article Title: Identification and validation of candidate genes dysregulated in alveolar macrophages of acute respiratory distress syndrome

doi: 10.7717/peerj.12312

Figure Lengend Snippet: (A) PPI network was constructed basing on the top 300 DEGs. The red icons represent up-regulated genes and the blue ones represent down-regulated genes. Furthermore, the connecting lines between the icons indicate potential interaction between different DEGs. (B) Module analysis of the DEGs enrolled in PPI network using MCODE method. The top five ranking clusters are shown according to MCODE score. (C) Functional enrichment analysis of genes in cluster 1. Likewise, the y axis and x axis represent enrichment terms and scores, respectively, and the color means p- value. (D) The top 10 hub genes are identified from PPI network using MCC method. The color represents MCC score. A darker red icon means higher MCC score and a lighter yellow one represents lower MCC score. (E) mRNA expression levels of CCL13, CCR2, FPR3 and PLAU in alveolar macrophages isolated from better prognosis subjects (VFDs > 12) and from poor prognosis individuals (VFDs <12).

Article Snippet: Rabbit polyclonal anti-human FPR3 antibody (orb608040, Biorbyt, UK), rabbit polyclonal anti-human CCR2 antibody (NBP1-48337, Novus Biologicals, USA) and HRP-conjugated secondary antibody (1:2000, Proteintech, IL, USA) were used.

Techniques: Construct, Functional Assay, Expressing, Isolation

Journal: PeerJ

Article Title: Identification and validation of candidate genes dysregulated in alveolar macrophages of acute respiratory distress syndrome

doi: 10.7717/peerj.12312

Figure Lengend Snippet: (A) Relative mRNA expressions of CCL13, CCR2, CX3CR1, CXCL16, FPR3 and PLAU on THP-1-derived macrophages in response to LPS were assessed using qRT-PCR. (B) The protein levels of CCR2 and FPR3 under a varying time of LPS treatment were evaluated by Western blot analysis. (C) CCR2 expression on surface of THP-1-derived macrophages after LPS treatment was detected by flow cytometry analysis. (D) Relative mRNA expressions of CCR2 and FPR3 on primary AMs collected from patients and volunteers were evaluated using qRT-PCR. (E) The protein levels of CCR2 and FPR3 in primary AMs were examined using Western blot assay. (F) CCR2 expression on surface of primary AMs was detected by flow cytometry analysis. Data are presented as mean ± SD and are representative of three independent experiments. ∗ P < 0.05, by unpaired, 2-tailed Student’s t test.

Article Snippet: Rabbit polyclonal anti-human FPR3 antibody (orb608040, Biorbyt, UK), rabbit polyclonal anti-human CCR2 antibody (NBP1-48337, Novus Biologicals, USA) and HRP-conjugated secondary antibody (1:2000, Proteintech, IL, USA) were used.

Techniques: Derivative Assay, Quantitative RT-PCR, Western Blot, Expressing, Flow Cytometry

Journal: PeerJ

Article Title: Identification and validation of candidate genes dysregulated in alveolar macrophages of acute respiratory distress syndrome

doi: 10.7717/peerj.12312

Figure Lengend Snippet: (A)Diagrammatic illustration for the chemotaxis assay procedure: Upper:1 ×10 4 of wild-type or siRNA-transfected macrophages were cultured in serum-free medium with or without LPS. Lower: Culture medium with or without CCL2/fMLP. (B) LPS-induced macrophage chemotaxis toward chemokine CCL2 was assessed using a transwell assay as described in the Methods. (C) Likewise, LPS-triggered macrophage migration toward fMLP was evaluated as above. (D) The correlation between CCR2 and FPR3 expression was analyzed using pearson correlation analysis with expression data of GSE89953 and GSE116560 . (E) Elevated protein expression of CCR2 induced by LPS stimulation was significantly attenuated by siRNA-FPR3 transfection. Data are presented as mean ± SD and are representative of 3 independent experiments. ∗ P < 0.05, by unpaired, two-tailed Student’s t test.

Article Snippet: Rabbit polyclonal anti-human FPR3 antibody (orb608040, Biorbyt, UK), rabbit polyclonal anti-human CCR2 antibody (NBP1-48337, Novus Biologicals, USA) and HRP-conjugated secondary antibody (1:2000, Proteintech, IL, USA) were used.

Techniques: Chemotaxis Assay, Transfection, Cell Culture, Transwell Assay, Migration, Expressing, Two Tailed Test

Journal: Nature

Article Title: FPR1 is the plague receptor on host immune cells

doi: 10.1038/s41586-019-1570-z

Figure Lengend Snippet: Human neutrophils were stained with the β-lactamase substrate CCF2-AM, infected with wild-type Y. pestis KIM D27 (WT, pMM83) or KLD29 (Δ lcrV , pMM83) variant defective for the type III secretion system (T3SS), and analyzed for ( a ) blue fluorescence (YopM-Bla translocation into neutrophils and CCF2-AM cleavage) or ( b ) green fluorescence to derive the percent of stained cells injected with T3SS effector. c , Inhibition of Y. pestis T3SS into human neutrophils by monoclonal (αFPR1m) and polyclonal antibodies against FPR1 (αFPR1p), bacterial LcrV, N-formylpeptide (fMLF), annexin A1 peptide, staphylococcal CHIPS and cyclosporin H. d , Differential interference contrast (DIC) and fluorescence microscopy of mock or fMLF treated differentiated U937 cells infected with green-fluorescent Y. pestis KIM D27 (pEGFP) and stained with F1-specific antibody (red) to reveal extracellular bacteria in merged images of mock, but not in merged images of fMLF-treated cells. Orange and green arrows point to extra- and intracellular bacteria, respectively. e , Antibodies and ligands of FPR1 inhibit Y. pestis T3SS of YopM-Bla into U937 macrophages. One of three repeats is shown ( a-e ). Error bars represent the s.e.m. (n = 3 biological replicates) ( c , e ). One-way ANOVA with Bonferroni Correction was used to identify significant differences ( c , e ): ***, P <0.001; **, P <0.01.

Article Snippet: The rabbit polyclonal antibodies targeting FPR1 and FPR2 were purchased from Thermo Fisher Scientific, while the rabbit polyclonal antibody to FPR3 was purchased from Abcam (Cambridge, Massachusetts, USA).

Techniques: Staining, Infection, Variant Assay, Fluorescence, Translocation Assay, Injection, Inhibition, Microscopy

Journal: Journal of Cancer

Article Title: Identification of crucial genes through WGCNA in the progression of gastric cancer

doi: 10.7150/jca.95757

Figure Lengend Snippet: The correlation between C1QA , C1QB , C1QC , FCER1G , FPR3 , TYROBP expression and the prognosis of gastric cancer was analyzed using GSE15459 cohort.

Article Snippet: C1QA monoclonal antibody (1:100, Cat.67063-1-Ig, Proteintech), C1QB rabbit pAb (1:100, Cat.382301, ZENBI0), C1QC polyclonal antibody (1:100, Cat.16889-1-AP, Proteintech), Anti- FPR3 (1:100, Cat.ab188785, abcam), DAP12 ( TYROBP ) rabbit pAb (1:100, Cat.383147, ZENBI0) and FCER1G (1:100, Cat.CQA4448, Cohesion) were used as primary antibodies, and working solution form Dako REALTM EnVisionTM test (Cat.K5007) were as secondary antibody.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: Identification of crucial genes through WGCNA in the progression of gastric cancer

doi: 10.7150/jca.95757

Figure Lengend Snippet: GSEA for samples with high hub gene expression and low hub gene expression. (A) The enriched gene sets in HALLMARK collection by samples of high C1QA , C1QB , C1QC , FCER1G , FPR3 , TYROBP expression, respectively. (B) Gene set enriched in the interferon gamma response ( p .adjust = 4.545e-10, NES =3.33, p -value = 1e-10). (C) Gene set enriched in the oxidative phosphorylation ( p .adjust = 4.545e-10, NES =-2.39, p -value = 1e-10). NES: normalized enrichment score. GSEA: Gene Set Enrichment Analysis.

Article Snippet: C1QA monoclonal antibody (1:100, Cat.67063-1-Ig, Proteintech), C1QB rabbit pAb (1:100, Cat.382301, ZENBI0), C1QC polyclonal antibody (1:100, Cat.16889-1-AP, Proteintech), Anti- FPR3 (1:100, Cat.ab188785, abcam), DAP12 ( TYROBP ) rabbit pAb (1:100, Cat.383147, ZENBI0) and FCER1G (1:100, Cat.CQA4448, Cohesion) were used as primary antibodies, and working solution form Dako REALTM EnVisionTM test (Cat.K5007) were as secondary antibody.

Techniques: Gene Expression, Expressing, Phospho-proteomics

Journal: Journal of Cancer

Article Title: Identification of crucial genes through WGCNA in the progression of gastric cancer

doi: 10.7150/jca.95757

Figure Lengend Snippet: Experimental verification of six hub genes in different gastric cancer stage tissues. (A) Statistical analysis of immunohistochemistry results in early-stage tissues (n = 9) and advanced stage tissues (n = 9). Data were assessed via the method of Mann-Whitney U test. * p -value < 0.05, ns, no significance. (B) Representative images of immunohistochemical staining for C1QA , C1QB , C1QC , FCER1G , FPR3 , and TYROBP between early stage and advanced stage gastric cancer patients. Scale bars = 100 μm.

Article Snippet: C1QA monoclonal antibody (1:100, Cat.67063-1-Ig, Proteintech), C1QB rabbit pAb (1:100, Cat.382301, ZENBI0), C1QC polyclonal antibody (1:100, Cat.16889-1-AP, Proteintech), Anti- FPR3 (1:100, Cat.ab188785, abcam), DAP12 ( TYROBP ) rabbit pAb (1:100, Cat.383147, ZENBI0) and FCER1G (1:100, Cat.CQA4448, Cohesion) were used as primary antibodies, and working solution form Dako REALTM EnVisionTM test (Cat.K5007) were as secondary antibody.

Techniques: Immunohistochemistry, MANN-WHITNEY, Immunohistochemical staining, Staining

Journal: Journal of Cancer

Article Title: Identification of crucial genes through WGCNA in the progression of gastric cancer

doi: 10.7150/jca.95757

Figure Lengend Snippet: The correlation between C1QA , C1QB , C1QC , FCER1G , FPR3 , TYROBP expression and the prognosis of gastric cancer was analyzed using GSE15459 cohort.

Article Snippet: C1QA monoclonal antibody (1:100, Cat.67063-1-Ig, Proteintech), C1QB rabbit pAb (1:100, Cat.382301, ZENBI0), C1QC polyclonal antibody (1:100, Cat.16889-1-AP, Proteintech), Anti- FPR3 (1:100, Cat.ab188785, abcam), DAP12 ( TYROBP ) rabbit pAb (1:100, Cat.383147, ZENBI0) and FCER1G (1:100, Cat.CQA4448, Cohesion) were used as primary antibodies, and working solution form Dako REALTM EnVisionTM test (Cat.K5007) were as secondary antibody.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: Identification of crucial genes through WGCNA in the progression of gastric cancer

doi: 10.7150/jca.95757

Figure Lengend Snippet: GSEA for samples with high hub gene expression and low hub gene expression. (A) The enriched gene sets in HALLMARK collection by samples of high C1QA , C1QB , C1QC , FCER1G , FPR3 , TYROBP expression, respectively. (B) Gene set enriched in the interferon gamma response ( p .adjust = 4.545e-10, NES =3.33, p -value = 1e-10). (C) Gene set enriched in the oxidative phosphorylation ( p .adjust = 4.545e-10, NES =-2.39, p -value = 1e-10). NES: normalized enrichment score. GSEA: Gene Set Enrichment Analysis.

Article Snippet: C1QA monoclonal antibody (1:100, Cat.67063-1-Ig, Proteintech), C1QB rabbit pAb (1:100, Cat.382301, ZENBI0), C1QC polyclonal antibody (1:100, Cat.16889-1-AP, Proteintech), Anti- FPR3 (1:100, Cat.ab188785, abcam), DAP12 ( TYROBP ) rabbit pAb (1:100, Cat.383147, ZENBI0) and FCER1G (1:100, Cat.CQA4448, Cohesion) were used as primary antibodies, and working solution form Dako REALTM EnVisionTM test (Cat.K5007) were as secondary antibody.

Techniques: Gene Expression, Expressing, Phospho-proteomics

Journal: Journal of Cancer

Article Title: Identification of crucial genes through WGCNA in the progression of gastric cancer

doi: 10.7150/jca.95757

Figure Lengend Snippet: Experimental verification of six hub genes in different gastric cancer stage tissues. (A) Statistical analysis of immunohistochemistry results in early-stage tissues (n = 9) and advanced stage tissues (n = 9). Data were assessed via the method of Mann-Whitney U test. * p -value < 0.05, ns, no significance. (B) Representative images of immunohistochemical staining for C1QA , C1QB , C1QC , FCER1G , FPR3 , and TYROBP between early stage and advanced stage gastric cancer patients. Scale bars = 100 μm.

Article Snippet: C1QA monoclonal antibody (1:100, Cat.67063-1-Ig, Proteintech), C1QB rabbit pAb (1:100, Cat.382301, ZENBI0), C1QC polyclonal antibody (1:100, Cat.16889-1-AP, Proteintech), Anti- FPR3 (1:100, Cat.ab188785, abcam), DAP12 ( TYROBP ) rabbit pAb (1:100, Cat.383147, ZENBI0) and FCER1G (1:100, Cat.CQA4448, Cohesion) were used as primary antibodies, and working solution form Dako REALTM EnVisionTM test (Cat.K5007) were as secondary antibody.

Techniques: Immunohistochemistry, MANN-WHITNEY, Immunohistochemical staining, Staining