Fig 2: Trail upregulation in aneurysm tissue of AngII-infused ApoE-/-Opg-/- mice.(A) Representative immunofluorescence images of aortic tissue sections from AngII-infused mice stained with anti-Trail (green) antibody. Scale bars indicate 100 µm. Areas selected by the white box (dotted line) in the Aneurysm group are magnified below. (B) Magnified immunofluorescence images of aortic tissue sections from the AngII-infused Aneurysm group stained with anti-Trail (green; a, f), anti-SMA (green; b, g), anti-vimentin (red; c, h), anti-Ki67 (red; d, i), and anti-F4/80 (red; e, j) antibodies in ApoE-/-Opg+/+ and ApoE-/-Opg-/- mice. Nuclei were stained with DAPI (blue; c-DAPI, h-DAPI). Scale bars indicate 25 µm. Medial layer (m), adventitial layer (a). The two arrowheads show borders of the medial layer. The white dotted line in the magnified panels indicates the border of the medial and adventitial layers. Small arrows in panel j indicate large round-shaped macrophages infiltrating the adventitia. Statistical significance: p<0.05. (C) Percent area of Trail expression in medial and adventitial layers of ApoE-/-Opg+/+ (n = 16) and ApoE-/-Opg-/- (n = 16) mice. Measurements for all samples are presented as box plots. Measurements for the three groups are presented as bar graphs. n = 4 and 8 for the No Aneurysm group, n = 5 and 5 for the Aneurysm group, and n = 7 and 3 for the Dissection group in ApoE-/-Opg+/+ and ApoE-/-Opg-/- mice, respectively. Statistical significance: p<0.05.

Fig 3: ISH staining of MGL mRNA in colon of healthy (a–c, e–g) and DSS-treated mice (d, h). MGL mRNA (FastRed) is seen in cells of the epithelium (ep) and the lamina propria (lp) (a, b), myenteric ganglia (casterisk), and in the circular (cm) and longitudinal (lm) muscle layer (c) of healthy colonic mucosa. In contrast, MGL gene expression drops throughout the whole colon after DSS treatment, particularly in the epithelium (ep) and the circular muscle layer (cm) (d, arrows). c and d Taken from ISH/IHC double staining of MGL mRNA (FastRed) and CD3 protein (purple, VIP). Immunofluorescence shows that MGL mRNA (red, FastRed) is abundantly stained in the circular (cm) as well as the longitudinal (lm; arrowheads) muscle layer (e). Co-localization of MGL mRNA with synaptophysin (green, Alexa 488) is rather low (yellow dots; arrows; e). There is only little MGL gene expression (pink, FastRed) in CD3+ cells (purple, VIP) (f). Gene expression of MGL in F4/80+ cells (purple, VIP) is decreased after DSS treatment (h) as compared to healthy animals (g). Quantification of MGL mRNA in F4/80+ cells in healthy versus diseased animals is shown in i; note the increased cell number in expression level 0 (no expression) and reduced number of cells in expression level 1. Calibration bar in a 20 µm; calibration bars in b, f, g, h 10 µm; calibration bars in c, d 50 µm; calibration bar in e 100 µm; cm circular muscle layer, ep epithelium, lp lamina propria, lm longitudinal muscle layer, cl crypt lumen, sm submucosa; *, myenteric ganglia. Arrows point at representative cells expressing MGL mRNA

Fig 4: Normal and aberrant complement C3 localization in Csmd1 wildtype versus knockout testes. a C3 mRNA expression in sexually immature whole testes, sexually mature whole testes, and whole ovary. RNA-seq of FACS-purified germ cell populations shows C3 expression decreases throughout spermatogenesis. Csmd1 expression data from Fig. 2 are rescaled and superimposed for ease of comparison. Line chart error bars reflect standard error of the mean. b Complement and macrophages are confined to the interstitial compartment in normal tubules. F4/80 immunofluorescence of whole mount tubule preparations shows abundance of macrophages along the interstitium (left). C3 immunofluorescence of tubule cross-sections shows expression in the interstitium, but not within the lumenal compartment (middle). Cross-section of downstream epididymis shows continued exclusion of C3 from the lumenal compartment (right). Individual tubules are circumscribed by dashed lines. Scale bars = 50 µm. c Boxplots depicting F4/80 abundance and C3 protein deposition in FACS-sorted Csmd1 wildtype vs. knockout testes. Both F4/80 and C3 are increased in Csmd1 knockout testes, though only significant for C3 (ANOVA p-value = 7.7 × 10-4). d RNA-seq of Csmd1 wildtype vs. knockout germ cell populations shows no significant difference in C3 mRNA expression (two-tailed t-test; p-value > 0.05). For all boxplots, center line = median; hinges = upper and lower quartiles; whiskers = 1.5 × interquartile range. Source data are provided as a Source Data file

Fig 5: ISH staining of CB2 mRNA in ileum of healthy (a, b, d, f) and LPS-treated (e, g) mice. Healthy mice show very low gene expression of CB2 (brown, DAB) in myenteric ganglia of the ileum (a marked by asterisk; arrows point at mRNA staining). Compared to myenteric ganglia, cells in lymph follicles show widespread CB2 gene expression, some located in CD3+ cells (b arrows). The CB2 gene is also expressed in lamina propria CD3+ cells of healthy animals, although at a low level (d arrows point at expression in representative cells). Cells often line up underneath the epithelium (ep; e). Also CD3 negative cells expressing the CB2 gene are visible In the lamina propria (lp) (e arrowhead). In addition, cells stained for F4/80 show CB2 mRNA in healthy (f) and LPS-treated (g) mice. There is only marginal expression of CB2 in epithelial cells even after LPS treatment (g; arrowhead). Quantification of CB2 mRNA levels in CD3+ cells is shown in c, indicating that CB2 gene expression is not significantly altered after LPS treatment. Calibration bars: 10 µm; ep epithelium, lp lamina propria, *, myenteric ganglion