Fig. 3

Overexpression of ERAP2 promotes CD4⁺ T cell pyroptosis. Normal CD4⁺ T cells were stimulated for 72 h. Then, CD4⁺ T cells were infected with control lentivirus or lentivirus encoding ERAP2 (LV-NC/ LV-ERAP2). A Flow cytometric analysis of CD4⁺ T cells treated as indicated and stained with Annexin V/7AAD (n = 3). Representative flow plots of Annexin V and 7-AAD staining of CD4⁺T cells and a histogram of the percentage of Annexin V⁺ 7AAD⁺ CD4⁺T cells are shown. B Concentrations of secreted IL-1β measured by ELISAs (n = 5). C Representative scatterplots of FLICA⁺ cells identified by flow cytometric analysis (n = 3). D T-cell death was quantified by measuring LDH release (n = 5). E Pyroptotic cells were identified by Hoechst 33,342/PI staining; the nuclei were stained blue with Hoechst 33,342, while pyroptotic cells were stained red with PI (n = 3). Scale bars: 50 μm. F Representative immunoblots showing ASC, NLRP3, cleaved Caspase-1, and GSDMD-N expression (n = 3). G Quantification of ASC, NLRP3, cleaved Caspase-1, and GSDMD-N protein expression shown in F (n = 3). All data are presented as the mean ± SD. Statistical significance was determined by Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NC, normal control; RA, rheumatoid arthritis