Figure 4. Transplantation of FREFs boosted Aire gene expression in the aged thymus and showed enhanced negative selection signaling strength via Nur77 in CD4^SP thymocytes of aged mice.

Same experimental setting as described in Figure 2. (A) Representative immunofluorescence staining images of Aire⁺ TECs (red) in K8⁺ TEC counterstaining (green). Data are representative of 3 biological replicates in each group with essentially identical results. Scale bar: 100 μm. (B) Summarized result shows the percentage area of Aire⁺ TECs against K8⁺ counterstaining based on the slides in A. Each symbol represents 1 thymic tissue section; 5–7 of these thymic tissue sections per thymus at different physical locations (nonsequential slides) were counted using 3 thymuses per group and analyzed with ImageJ software (NIH; a total of 17–19 thymic tissue sections from 3 individual thymuses per group were observed). (C) Flow cytometric results show increased Nur77 signaling strength (relative quantitative [RQ] mean fluorescence intensity [MFI]) in CD4^SP thymocytes of young (control) or aged mice that were engrafted with MEFs or 2 types of FREFs. Left panel: histogram of Nur77 MFI in CD4^SP thymocytes; right panel: Nur77 RQ-MFI in CD4^SP populations of various groups (n = 7–8 mice per group). All results represent the mean ± SD. The statistical analysis was performed by 1-way Newman-Keuls multiple-comparisons test. **P < 0.01, ***P < 0.001. n.s., not significant.