Figure 6.

Cell internalization of ASC-Exo in cultured monocytes and dermal fibroblasts (DFb) of diabetic mice and role of ASC-Exo in Smad3-dependent TGF-β stimulation of ECM production. (A) Cellular uptake of ASC-Exo in cultured monocytes by confocal microscopy demonstrated rapid uptake of ASC-Exo. Merged images of DAPI-labelled nucleus (blue), R18-labelled ASC-Exo (red), and phalloidin-labelled cytoskeleton (green) revealed localization of ASC-Exo around the cells at 2 h and into the cells at 12 h. (B) ASC-Exo uptake by cultured dermal fibroblasts. (C) ASC-Exo treated monocytes and dermal fibroblasts increase the production TGF-β1. TGF-β1 levels were measured using enzyme-linked immunosorbent assay (** p < 0.01). Data were expressed in means ± SE, n = 3/group, Student’s t-tests. (D) Experimental design of SIS3, a specific inhibitor of Smad3, on TGF-β1 dependent Smad3 phosphorylation and extracellular matrix production in cultured DFb. The DFb cultures were serum-starved for 24 h and treated with 3 µM SIS3 for 1 h, and then supernatant of ASC-Exo-treated monocyte culture was added. After 48 h, the conditioned media were subjected to immunoblotting with antibodies for Col-I, α-SMA, p-Smad3, and β-actin, respectively. One representative of four independent experiments is shown. Error bars are mean ± SE. Data were analyzed using independent unpaired two-tailed Student’s t-tests or 1-way ANOVA with Bonferroni correction. (E–H) SIS3 inhibited the protein expression of Col-I, α-SMA, and p-Smad3 induced by supernatant of ASC-Exo-treated monocyte culture in dermal fibroblasts. Data are expressed as the mean ± SE of four independent experiments. *** p < 0.001 compared with the value in the supernatant of ASC-Exo-treated monocyte culture treated DFb without SIS3.