Fig. 2

ERAP2 regulates pyroptosis in CD4⁺ T cells. NC and RA CD4⁺ T cells were stimulated for 72 h. Then, NC CD4⁺ T cells were infected with control shRNA virus (NC-shcontrol), and RA CD4⁺ T cells were infected with control shRNA virus or ERAP2 shRNA virus (RA-shcontrol/ RA-shERAP2). A, B ERAP2 expression in isolated CD4⁺ T cells is shown in representative immunoblots (left) and plots of the relative band density for NCs (n = 5) and RA patients (n = 5) with normalization to β-actin expression (right). C ERAP2 transcript levels in NC and RA CD4⁺ T cells (n = 5). D, E 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 7AAD staining of CD4⁺ T cells and a histogram of the percentage of Annexin V⁺ 7AAD⁺ CD4⁺ T cells are shown, and representative scatterplots of FLICA⁺ cells identified by flow cytometric analysis (n = 3). F Concentrations of secreted IL-1β measured by ELISAs (n = 5). G, H 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. I T-cell death was quantified by measuring LDH release (n = 5). J Representative immunoblots showing ASC, NLRP3, cleaved Caspase-1, and GSDMD-N expression (n = 3). K Quantification of ASC, NLRP3, cleaved Caspase-1, and GSDMD-N protein expression shown in J (n = 3). All data are shown as the mean ± SD. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple-comparisons test for multigroup comparisons or Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NC, normal control; RA, rheumatoid arthritis