Fig 1: a Experimental design. EVs: Extracellular Vesicles b Scatterplot showing the somatic cell count (SCC) of the Control group, the Low SCC and the High SCC on a logarithmic scale. Lower-case letters denote significant differences (P < 0.05) after applying non-parametric Kruskal-Wallis and Dunn’s multiple comparisons tests. c Diameter distribution of milk cells in one High SCC (red), Low SCC (yellow) and Control (Green) sample, respectively. d Transmission electron microscopy (TEM) observation of isolated milk EVs. Black arrows indicate microvesicles and exosomes; white ovals highlight non-vesicles and white arrows point at shapeless aggregations with low electron density [29]. e Representative graphs of size distribution from three milk EVs samples measured with tunable resistive pulse sensing technology. f Western blot characterization for the EVs protein markers CXN, TSG101, CD81, CD9 and MFGE8. The absence of CXN suggests no contamination during the isolation with intracellular debris; L: Ladder; 1-9: Milk EVs pellets; MG: Mammary gland tissue; MF: Milk Fat; MC: Milk cells. Full-length blots are presented in Supplementary Fig. S4
Fig 2: Extracellular medin deposition and aggregation is mediated by VSMC EVs. (a) Western blot of VSMC small (sEVs), medium (mEVs), large (lEVs) extracellular vesicles (EVs) and whole cell lysate (WCL). CD63 was used as a sEV marker and calnexin was used as a cell marker. (b) Slot blot of EVs and quantification showing surface localization of medin and MFG‐E8. TSG101 was used as a control which localises mainly inside the lumen of EVs, while CD63 was used as a surface protein control (n = 3 from 35‐year‐old female (35F)). (c) Immunofluorescence for medin and CD63 in decellularized ECM. Scale bar 25 μm. (d) Super‐resolution microscopy of (1) larger medin and EV aggregates, (2) individual sEVs with medin on the surface and (3) individual sEVs which do not colocalize with medin. (e) Immunofluorescent staining and quantification of ECM synthesized with or without EV secretion inhibitor 3‐O‐methylsphingomyelin (3‐OMS). Grey data points represent deposits within a field of view and black points represent average from each donor. Scale bar 25 μm, (n = 6 from 35F, 22‐year‐old male (22), 20‐year‐old male (20 M)). Unpaired Student's t test, ***p < 0.005, ****p < 0.001. (f) Thioflavin T (ThT) aggregation assay and quantification of recombinant medin peptide with or without sEVs added (n = 8–9 from 35F). Unpaired Student's t test, **p < 0.01. All data is displayed as mean ± SD and was tested for normality using Shapiro–Wilk test.
Fig 3: Vascular smooth muscle cell senescence enhances extracellular medin accumulation. (a) Immunohistochemistry of medin in the medial layer of aorta from human subjects. The black arrows highlight areas surrounding the nuclei which are not stained. (b) Correlation of medin staining with age (n = 25). Spearman correlation. (c) Representative Western blotting for medin and MFG‐E8 in early (EP) and late (LP) passage ECM. Fibronectin (FN) and Coomassie used as loading controls. (d) Slot blot for medin and quantification in EP ECM and LP ECM (n = 6 from 35‐year‐old female (35F), 22‐year‐old male (22 M), 20‐year‐old male (20 M)). Unpaired Student's t test, **p < 0.01. (e) Immunofluorescence of medin deposition in fibril‐like form (yellow arrow) in the LP ECM and colocalization with CD63 (white arrow) and quantification of medin deposition area and percentage of medin in fibril‐like form (n = 6 from 35F, 22 M, 20 M). Grey data points represent individual fields of view. Black data points represent average from each donor. Unpaired Student's t test, ****p < 0.001. Scale bar 25 μm. (f) Super resolution microscopy of medin in the LP ECM colocalized with CD63 (white arrows). All data is displayed as mean ± SD and was tested for normality using Shapiro–Wilk test.
Supplier Page from MilliporeSigma for Anti-MFGE8 antibody produced in rabbit