Fig. 3: AIRE dysregulation in thymus of 3-month-old mdx mice.

Representative confocal microscope images (left) and tile scan reconstruction (right) of thymic lobes from 3-month-old C57Bl and mdx mice. Despite a comparable AIRE+ cell pattern distribution embedded within CK5+ thymic medulla of both mice, in mdx thymus immunofluorescence staining for AIRE appeared less evident. Nuclei were counterstained with DAPI (a). Representative images of western blot analysis showing the expression of the AIRE and SIRT-1 proteins in the thymus of C57Bl and mdx mice. Densitometric analyses are shown as the AIRE/vinculin ratio and SIRT-1/actin ratio (b). For the identification and sorting of cTEC and mTEC from C57bl and mdx mice, stained thymus cell suspension was analysed using flow cytometry. Representative FACS profile are shown. The numbers within the panels indicate the percentage of each population of live cells, a gate of CD45-negative and EpCAM-positive events represents TEC cells. Within the TEC gate, two population are separated by level of Ly5.1 and UEA-1. MHC II molecules were highly expressed on cTECs (c). Following FACS isolation of mTEC and cTEC, RT-qPCR experiments showed the downregulation of AIRE and Fezf2 in isolated mdx mTEC related to mTEC from C57Bl thymus (d). The expression in thymus of AIRE-regulators RANK and CD40 was similar between mTEC isolated from C57Bl and mdx mice (e). RT-qPCR revealed diminished expression of AIRE-dependent genes Ins2 and Spt1 in dystrophic mTEC and in control cTEC related to mTEC isolated from C57Bl thymus (f). Scale bar: 50 μm (a). The comparisons between the averages of the groups were evaluated using two-sided Student’s t-test. b ****p < 0.0001 (AIRE), **p = 0.0050 (SIRT-1). d p = 0.0436 (AIRE) and p = 0.0220 (fezf2) in dystrophic mTECs vs C57Bl mTECs; p = 0.0224 (AIRE) and p = 0.00089 (fezf2) in C57Bl mTECs vs C57Bl cTECs. f p = 0.0413 (Ins-2) in mdx mTECs vs C57Bl mTECs; p = 0.0017 (Ins-2), p = 0.0178 (spt1), p = 0.0413 (mup4), p = 0.0271 (s100a8) in C57Bl mTECs vs C57Bl cTECs; p = 0.0210 (mup4) in C57Bl mTECs vs C57Bl cTECs. Data are presented as mean ± SD of three independent experiments with n = 6 mice/group (a, b); n = 8 (c); n = 8 mice divided into two independent groups examined in n = 2 independent experiments (d–f). Source data are provided as a Source Data file.