Fig 2: Thbs1R1034C-mutant mice show accumulation of THBS1 in TM.(A) THBS1-stained cryosections of the iridocorneal angle region from 4-month-old Thbs1R1034C homozygous mice and age-matched controls. TM was localized adjacent to the CD31-stained SC. Thbs1R1034C-mutant mice showed increased THBS1 protein expression in TM compared with WT controls. Nuclei were stained with DAPI. Scale bar: 100 µm; insets, ×2.4. (B) IF staining for THBS1 in corneal whole mounts showing an en face view of protein localization. SC is indicated by dashed lines, as revealed by CD31 staining. Scale bar: 200 µm; insets, ×0.23 (upper left in B and C). (C) Progressive THBS1 protein accumulation in TM was evident in homozygous mice, and a similar pattern of accumulated THBS1 protein was evident in ThbsR1034C heterozygous mice. Scale bar: 200 µm; insets, ×3.6 (lower right in B and C). (D) Quantification of THBS1 in TM based on fluorescence intensity (100 µm2 fields were captured for intensity measurements; n = 9 for each time point). AU, arbitrary fluorescence units. Data represent the mean ± SEM. (E) Thbs1R1034C homozygous mice did not show increased Thbs1 mRNA expression (as determined by unpaired 2-tailed Student’s t test). Relative mRNA levels were normalized to Gapdh. n = 3. Data represent the mean ± SEM. (F) Western blot of protein extracts from the corneal limbal ring containing the iridocorneal angle region. Higher levels of THBS1 protein were detected in homozygous Thbs1R1034C mice, however, there was no evidence of ER stress in the mutant mice at age 16 months of age compared with age-matched controls based on the expression of GRP94, IRE1A, and BIP.

Fig 3: Mouse milk is rich in extracellular vesicles.a) Transmission electron microscopy (TEM) images of EVs isolated from mouse and human milk. b) According to nanoparticle tracking analysis, human milk contains an order of magnitude more EVs than mouse milk. c) There is no significant difference in the size of EVs from human and mouse milk as measured by nanoparticle tracking analysis. d) Western blot characterization shows that mouse and human milk EVs express the EV markers CD9 and TGS101, but do not express Grp94, a protein from the endoplasmic reticulum which should not be present in samples of EVs. e) Nanoparticle tracking analysis showing the size distribution of EVs in mouse and human milk.

Fig 4: Characterisation of extracellular vesicles isolated from BeWo cells and HTR8/SVneo. Top: experimental design. NTA was performed in the three different pellets (2000, 10,000 and 100,000 g) isolated from (a) BeWo cells, and (b) HTR8/SVneo, control and high D‐glucose treatment. As part of the characterisation, TEM images were obtained for the same samples, (c) BeWo cells and (d) HTR8/Svneo. Western blot was performed to evaluate the expression of extracellular vesicle markers CD9 and TSG101, specific placental marker PLAP and cell control marker Grp94 using samples from (e) BeWo cells and (f) HTR8/SVneo.