Journal: Frontiers in physiology

Article Title: Potential biomarkers and immune cell infiltration involved in aortic valve calcification identified through integrated bioinformatics analysis.

doi: 10.3389/fphys.2022.944551

Figure Lengend Snippet: FIGURE 5 Validation of key genes SCG2 and CCL19. (A,B) Validation of the expression and receiver operating characteristic (ROC) curve of SCG2 and CCL19 in the merged metadata cohort. (C,D) Validation of the expression of SCG2 and CCL19 and ROC curve for evaluating the discriminatory accuracy of SCG2 and CCL19 in the validation dataset GSE83453. (E,F) GSEA analysis result show the top ten enrichment terms (GO and KEGG respectively) of regrouped high SCG2 expression group. (G,H) GSEA analysis result show the top ten enrichment terms (GO and KEGG respectively) of regrouped high CCL19 expression group. **p < 0.01, ****p < 0.0001.

Article Snippet: Sections were deparaffinated, blocked, and incubated with the primary anti-SCG2 antibody (Proteintech, 20357-1-AP) or Anti-CCL19 Antibody (Proteintech, 13397-1-AP) at 4°C overnight.

Techniques: Biomarker Discovery, Expressing

Journal: Frontiers in physiology

Article Title: Potential biomarkers and immune cell infiltration involved in aortic valve calcification identified through integrated bioinformatics analysis.

doi: 10.3389/fphys.2022.944551

Figure Lengend Snippet: FIGURE 7 Correlation analysis between key genes and immune cells. (A) Correlation analysis for SCG2. (B) Correlation analysis for CCL19. Size of the dots represents the strength of the correlation between genes and immune cells, and color of the dots represents p-value. p < 0.05 was considered statistically significant. (C,D) Scatter plot showing the correlation between SCG2/CCL19 and immune cells. The X-axis represents the mRNA expression level of genes, the Y-axis represents the expression abundance of immune cells, and the R value represents the correlation coefficient.

Article Snippet: Sections were deparaffinated, blocked, and incubated with the primary anti-SCG2 antibody (Proteintech, 20357-1-AP) or Anti-CCL19 Antibody (Proteintech, 13397-1-AP) at 4°C overnight.

Techniques: Expressing

Journal: Frontiers in physiology

Article Title: Potential biomarkers and immune cell infiltration involved in aortic valve calcification identified through integrated bioinformatics analysis.

doi: 10.3389/fphys.2022.944551

Figure Lengend Snippet: FIGURE 8 Validation at single cell level and tissue level. (A) tSNE visualization of clustering revealed 12 cell populations. Cell population identities were determined based on marker gene expression. (B) Heatmap of top 5 marker genes for each cell cluster. (C) Heatmap shows the expression of SCG2 and CCL19 in 12 clusters of cells in CAVD group and normal control group, respectively. The transition from blue to orange represents the increased expression level. (D) The heatmap of GSEA analysis results shows the hallmark functional pathways enriched in each of the calcified or normal aortic valve cell clusters. The transition from blue to red indicates the increased enrichment of pathways. (E) H and E staining and immunohistochemical staining of human aortic valve tissue sections. Macroscopic and micrographs views of immunohistochemical staining of SCG2 and CCL19 from left to right. (F) Histograms of quantitative immunohistochemical staining results. The expression of SCG2 and CCL19 was significantly increased in CAVD group. AUC: Area under the ROC curve; 95% CI: 95% confidence interval; Norm: normal; CAVD: calcific aortic valve disease; VICs: valvular interstitial cells; VECs: valvular endothelial cell; TCs: T cells. ***p < 0.001.

Article Snippet: Sections were deparaffinated, blocked, and incubated with the primary anti-SCG2 antibody (Proteintech, 20357-1-AP) or Anti-CCL19 Antibody (Proteintech, 13397-1-AP) at 4°C overnight.

Techniques: Biomarker Discovery, Marker, Gene Expression, Expressing, Control, Functional Assay, Staining, Immunohistochemical staining

Journal: bioRxiv

Article Title: Single-cell transcriptomes of the aging human skin reveal loss of fibroblast priming

doi: 10.1101/633131

Figure Lengend Snippet: t-SNE and violin plots showing population-specific expression levels for the secretory-reticular and pro-inflammatory markers CCL19 (A) and TSPAN8 (B), respectively. In the t-SNE plots, red indicates maximum relative expression and blue indicates low or no expression of a particular gene. In the violin plots, the X-axis depicts cell cluster identity while the Y-axis represents gene expression in log(TPM). (C,D) Representative confocal images of human skin sections stained for CCL19 (red, C) TSPAN8 (green, D) and VIM (grey). Papillary and reticular (superficial and deep) dermal regions are indicated. Nuclei were stained with DAPI (blue). Dotted lines denote boundaries between papillary and reticular dermis. Images are shown at 40× original magnification. Scale bar, 50 μm. TPM: transcripts per kilobase million.

Article Snippet: Primary antibodies used were rabbit anti-Tspan8 (Abcam, Ab230448, 1:200), mouse anti-Ccl19 (R&D Systems, MAB361, 15 µg/ml), rabbit anti-Vimentin (Cell Signaling, D21H3, 1:100) and chicken anti-Vimentin (Abcam, Ab24525, 1:2000).

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: Lymphotoxin-alpha expression in the meninges causes lymphoid tissue formation and neurodegeneration

doi: 10.1101/2021.04.27.441396

Figure Lengend Snippet: (A) Primary meningeal cells were treated with 100ng LTα for 24 hours and changes in gene expression measured by RT-PCR. Ratio changes calculated for LTα treated over non-treated showed increased gene expression levels for CCL19, CXCL13 and LTBR. * p<0.05. (B) The cortical meninges were microdissected from 4 x 10µm section from 28 dpi GFP or LVLTα injected animals for RNA extraction. Changes in mRNA levels for chemokines and receptors was measured by RT-PCR and shown here as Cytokine treated compared to GFP animals. * p<0.05 t-test on ΔΔCt values. (C) In the dissected meninges, CXCL13 transcript showed the greatest fold change in cytokine versus GFP animals at 28 dpi. ** p<0.01. (D) Immunostaining for CXCL13 at 28 dpi showed expression on blood vessels and lymphatic channels and on a network of cell processes through the immune cell infiltrate. Scale bar = 100µm. (E) Higher magnification shows CXCL13+ staining on cells with follicular dendritic cell morphology with multiple thin processes. Scale bar = 40µm. (F) IHC staining against ED5, a marker for follicular dendritic cells showed a dense network of cells through the infiltrates at 28 dpi. Inset box shows a higher magnification view. (G) CXCL13/CXCR5 double immunofluorescence and CD79a immunostaining on a serial section of cortical surface infiltrates. (H) Triple immunostaining for CCL21 and CXCL13 in regions with CD3+ T-cells showed distinct localisation patterns. (I) CXCL12 immunostaining was seen throughout the aggregates, together with expression of its receptor CXCR4 receptor (J) . CCL21 and CCL19 double immunofluorescence in midline (K) and cortical aggregates (L) . (M) Cells expressing CCR7 were found throughout the midline and cortical aggregates.

Article Snippet: The primary antibodies used were: mouse anti-MOG (clone Y10, Prof Reynolds, Imperial College London, UK); rabbit anti-myelin basic protein (MBP) (Polyclonal, Merck, Darmstadt, Germany); mouse anti-neurofilament-H protein (clone NE14; Merck, Darmstadt, Germany); rabbit anti-IBA1 (Polyclonal IgG, Wako Pure Chemical Corporation, USA); rabbit anti-Glial Fibrillary Acidic Protein (GFAP) (Polyclonal, Dako Agilent, Santa Clara, CA, USA); mouse anti-NeuN (clone A60, Merck, Darmstadt, Germany); mouse anti-CD79a (ThermoFisher); mouse anti-CD4 (AbD Serotec); mouse anti-CD8 (AbD Serotec) goat anti-human LTA (R&D systems); rabbit anti-laminin (Wako, Japan); rabbit anti-cleaved caspase-3 (Cell signalling, USA); goat anti-CXCL13 (R&D systems); mouse anti-CCL21 (R&D systems); mouse anti-CCL19 (R&D systems); mouse anti-CXCL12 (monoclonal, Sigma UK); rabbit anti-CXCR4 (Sigma, UK); goat anti-LTBR (polyclonal Goat, R&D); rabbit anti-Ki67 (Santa-Cruz); rabbit anti-CD3 (DAKO); rabbit anti-IgG (R&D); rabbit anti-laminin (Novusbio); mouse anti-MadCam (monoclonal mouse, R&D systems); mouser anti-CNPase (Millipore); rabbit anti-CXCR5 (Abcam, UK), rabbit anti-podoplanin (Abcam, UK); mouse anti-CCR7 (mouse monoclonal, R&D systems).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Injection, RNA Extraction, Immunostaining, Staining, Immunohistochemistry, Marker, Immunofluorescence, Triple Immunostaining