Fig 2: Neu1-/- exosomes contain increased levels of components of the TGF-ß and WNT signaling pathways.(A) Exosomes exocytosed by Neu1-/- myofibroblasts were verified on immunoblots probed with antibodies against established exosomal markers [Alix, CD81, CD9, flottilin-1 (Flot1), Syndecan-1 (SDC1), and syntenin 1 (SDCBP1)]. (B) Immunoblot analyses of WT and Neu1-/- exosomes performed with antibodies against canonical (ß-catenin, GSK3ß, LRP5, and WNT3a) and noncanonical (WNT5a/b) WNT signaling. (C and D) Immunoblots of WT and Neu1-/- exosomes probed with anti–TGF-ß1 (C) and LAP (D) antibodies. (E) Exosomes treated with proteinase K were analyzed on immunoblots probed with anti–TGF-ß1, anti-LAP, anti-WNT5a/b, and anti–ß-catenin antibodies.

Fig 3: Characterization of human MoDC exo subtypes. (A) NTA revealed correct size range of MoDCexos of ~150 nm. (B) Immunoblot revealing shared and distinct proteins in MoDC exo subtypes, including exosomal markers C81, TSG101, and CD9; anti-inflammatory cytokine TGFB1; and proinflammatory cytokines IL6, IL1B, and TNFa. (C) SEM images showing cup-shaped MoDC exos; (D) immunogold TEM images of regMoDC exos revealing intraluminal and surface localization of (D) TGFB1 and (E) IL10, as well as (F) surface expression of exosomal marker CD63 (arrows).

Fig 4: Recipient MoDCs take up exos, altering inflammatory cytokine profiles. (A) Dil (red)-labeled MoDC exos (arrows) inside recipient MoDCs counterstained with DAPI nuclear stain (blue), and phalloidin (green) for the cell membrane and visualized using confocal microscopy (upper panel images captured with 20× lens and lower panel with 63× lens). (B) Colocalization of TGFB1 in MoDCs +/- regMoDCexo or iMoDCexo treatment using immunogold TEM. (C) Q-rt-PCR through TaqMan gene expression using delta-delta CT and plotted as relative fold change of inflammatory cytokines IL-6, IL-10, IL-1B, TNFa, and IL-23.