Journal: Journal of Inflammation Research

Article Title: APOE and CCR2 : Potential Macrophage-Specific Biomarkers in the Rheumatoid Arthritis Synovial Microenvironment Identified by Bioinformatics and Experimental Verification in Murine Models

doi: 10.2147/JIR.S587712

Figure Lengend Snippet: Preprocessing of single-cell RNA sequencing (scRNA-seq) dataset and identification of synovial cell in GSE192504 database. ( A and B ) Highly variable genes filtering and PCA clustering of scRNA-seq data on the synovial cells isolated from the collagen-induced arthritis (CIA) mice group and the healthy control mice (normal) group. ( C and D ) Annotations and the proportions of different cell types between the CIA and normal group. Monocytes, macrophages, and fibroblasts accounted for significant proportions of synovial cells in the CIA group compared to the normal group. ( E ) t-SNE projections and cell annotation of scRNA-seq data extracted from the CIA group. Synovial cells mainly included monocytes, macrophages, and fibroblast-like synoviocytes in the CIA group. Cell types were annotated based on canonical marker gene expression (see Methods). ( F ) The expression levels and distribution of 10 hub genes in different synovial cells of the CIA group. Violin plots and t-SNE plots both showed specifical high expression of APOE and CCR2 in macrophages.

Article Snippet: The sections were then blocked with 3% bovine serum albumin in phosphate-buffered saline (PBS) for 30 min, followed by overnight incubation at 4 °C with the following primary antibodies (1:100 dilution): anti-apolipoprotein E (APOE) (rabbit monoclonal antibody, Proteintech Group, Inc., China), anti-placenta specific 8 (PLAC8) (rabbit monoclonal antibody, Proteintech Group, Inc., China), anti-CD74 (mouse monoclonal antibody, Proteintech Group, Inc., China), anti-podoplanin (PDPN) (mouse monoclonal antibody, Proteintech Group, Inc., China).

Techniques: Single Cell, RNA Sequencing, Isolation, Control, Marker, Gene Expression, Expressing

Journal: Journal of Inflammation Research

Article Title: APOE and CCR2 : Potential Macrophage-Specific Biomarkers in the Rheumatoid Arthritis Synovial Microenvironment Identified by Bioinformatics and Experimental Verification in Murine Models

doi: 10.2147/JIR.S587712

Figure Lengend Snippet: Correlation analysis between two hub gene expressions and GSVA immune-enrichment scores. ( A and B ) The heatmaps showed APOE and CCR2 expression and functional enrichment scores of RA patients in the GSE48780 database. The Spearman R and P values were respectively visualized by using the right bar graphs and line graphs.

Article Snippet: The sections were then blocked with 3% bovine serum albumin in phosphate-buffered saline (PBS) for 30 min, followed by overnight incubation at 4 °C with the following primary antibodies (1:100 dilution): anti-apolipoprotein E (APOE) (rabbit monoclonal antibody, Proteintech Group, Inc., China), anti-placenta specific 8 (PLAC8) (rabbit monoclonal antibody, Proteintech Group, Inc., China), anti-CD74 (mouse monoclonal antibody, Proteintech Group, Inc., China), anti-podoplanin (PDPN) (mouse monoclonal antibody, Proteintech Group, Inc., China).

Techniques: Expressing, Functional Assay

Journal: Journal of Inflammation Research

Article Title: APOE and CCR2 : Potential Macrophage-Specific Biomarkers in the Rheumatoid Arthritis Synovial Microenvironment Identified by Bioinformatics and Experimental Verification in Murine Models

doi: 10.2147/JIR.S587712

Figure Lengend Snippet: Changes in each macrophage cluster upon stimulation with LPS/IFN-γ or IL-4. ( A ) Representative images of immunofluorescence of each macrophage cluster. Blue represented the genetic material stained with DAPI and green showed fluorescence of APOE, PDPN, CD74, and PLAC8, which are regarded as specific protein markers of macrophage clusters. ( B ) Fluorescence intensity of APOE, PDPN, CD74, and PLAC8. Data are shown as mean ± SD from three independent biological experiments. * P < 0.05; *** P < 0.001.

Article Snippet: The sections were then blocked with 3% bovine serum albumin in phosphate-buffered saline (PBS) for 30 min, followed by overnight incubation at 4 °C with the following primary antibodies (1:100 dilution): anti-apolipoprotein E (APOE) (rabbit monoclonal antibody, Proteintech Group, Inc., China), anti-placenta specific 8 (PLAC8) (rabbit monoclonal antibody, Proteintech Group, Inc., China), anti-CD74 (mouse monoclonal antibody, Proteintech Group, Inc., China), anti-podoplanin (PDPN) (mouse monoclonal antibody, Proteintech Group, Inc., China).

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Journal of Inflammation Research

Article Title: APOE and CCR2 : Potential Macrophage-Specific Biomarkers in the Rheumatoid Arthritis Synovial Microenvironment Identified by Bioinformatics and Experimental Verification in Murine Models

doi: 10.2147/JIR.S587712

Figure Lengend Snippet: Gene and protein expression levels of APOE and CCR2 in LPS/IFN-γ or IL-4-stimulated RAW 264.7 cells. ( A ) Relative gene expression levels of APOE and CCR2 at 12h and 24 h after the addition of LPS/IFN-γ or IL-4. ( B ) APOE and CCR2 protein levels analyzed by ELISA at 24h upon stimulation with LPS/IFN-γ or IL-4. Data are shown as mean ± SD from three independent biological experiments. ** P < 0.01; *** P < 0.001.

Article Snippet: The sections were then blocked with 3% bovine serum albumin in phosphate-buffered saline (PBS) for 30 min, followed by overnight incubation at 4 °C with the following primary antibodies (1:100 dilution): anti-apolipoprotein E (APOE) (rabbit monoclonal antibody, Proteintech Group, Inc., China), anti-placenta specific 8 (PLAC8) (rabbit monoclonal antibody, Proteintech Group, Inc., China), anti-CD74 (mouse monoclonal antibody, Proteintech Group, Inc., China), anti-podoplanin (PDPN) (mouse monoclonal antibody, Proteintech Group, Inc., China).

Techniques: Expressing, Gene Expression, Enzyme-linked Immunosorbent Assay