Fig. 1: Altered thymic architecture in mdx mice.

Representative images of H&E staining of thymus of 3-month-old C57Bl and mdx mice revealed differences in medullary/cortex boundaries between animals (dashed white line) (a). WB analysis showed the downregulation of cytokeratin 14/16 in mdx thymus related to C57Bl (b). Thymic architecture of C57Bl and mdx mice characterized by immunofluorescence staining for cortical cytokeratin CK8 (green) and medullary cytokeratin CK5 (red) confirmed changes in dystrophic thymic environment. Graph displays fluorescence area % occupied by CK5 and CK8, as calculated by ImageJ software (c). Double immunofluorescence staining for CK5 (red) and CD3 (green) of C57Bl and mdx thymi portrayed a loosen embedding of CD3+ cells within dystrophic medulla. d Staining of ghrelin (GHR) and ghrelin receptor (GHS-R) (red) showed a comparable distribution of GHR between animals, but a prevalent expression of GHS-R in thymus of C57Bl mice. Of note, GHR was preferentially found in proximity of cortical CK8 (green). e Graph displays fluorescence area % occupied by GHS-R, as calculated by ImageJ software, in C57Bl and mdx mice (f). Expression of FoxP3+ cells (magenta) was evaluated by immunofluorescence staining within CK5+ thymic medulla (green). C-terminal containing dystrophin isoforms were detected by DYS-2 antibody (red) to identify a specific protein distribution within thymus. mTEC maturation level was evaluated by immunohistochemistry staining of C57Bl and mdx thyme with UEA-1. For fluorescence microscopy, nuclei were counterstained with DAPI (g). Scale bars: 200 μm (a); 100 μm for confocal tile scan reconstruction (left) and 20 μm for higher magnification confocal microscope images (right) (c, e); 50 μm (d); 50 μm (left) and 10 μm for higher magnification confocal microscope images (right) (g). The comparisons between the averages of the two groups were evaluated using two-sided Student’s t-test. b *p = 0.0495. c ***p = 0.0007. f **p = 0.0046. Data are presented as mean ± SD of three independent experiments with n = 6 mice/group. For Ck5–Ck8 immunofluorescence staining n = 12 images/mice have been quantified, GHS-R staining was quantified in n = 12 and n = 8 images of C57Bl and mdx mice, respectively. Source data are provided as a Source Data file.