(A) Heatmap displaying the differential expression patterns of 1,913 DEGs in CD14⁺ macrophages from RA-SF (n = 6) compared with normal macrophages (n = 2) differentiated from peripheral CD14⁺ monocytes. Red and green colors denote upregulation and downregulation, respectively. Mφ, macrophages. (B) Top 10 TFs enriched by up- and downregulated DEGs in RA synovial macrophages. (C) Venn diagram depicting the overlap between DEGs in synovial macrophages from RA patients and those in NFAT5-deficient RA synovial fibroblasts and human endothelial cells. P value indicates the significance of 302 overlapping DEGs, including 176 upregulated and 126 downregulated DEGs. Empirical hypothesis testing based on 100,000 random permutations was performed to determine the P value of the overlapping DEGs. (D) Cellular processes enriched by the DEGs in synovial macrophages from RA patients. Heatmap with violet color gradient represents the level of significance for each cellular process. Dark violet and bright violet refer to high (P < 0.01) and moderate (P < 0.05) enrichment, respectivel. The P values were determined by DAVID software using a hypergeometric test method. The bar graph on the right depicts the percentages of overlap between the DEGs associated with each cellular process and NFAT5 target genes. (E) Enhanced NFAT5 expression in RA synovial macrophages. Synovial macrophages were freshly isolated from synovial fluid of RA patients using anti-CD14 magnetic beads. Normal macrophages were differentiated from peripheral monocytes of healthy subjects. NFAT5 expression was determined by real-time PCR (n = 9), Western blot, and flow cytometric analyses. The gray plot in the flow cytometric graph indicates the isotype control in normal macrophages. NFAT5 mRNA expression is shown as a the fold increase relative to GAPDH mRNA. Data represent the mean ± SD. **P < 0.005 versus normal macrophages, by Mann-Whitney U test.