Figure 1
Apoptotic cell-derived EVs promote TGFβ in macrophages in vitro. (a) Western blotting of EVs markers: Total cell lysate (Total cell) and EVs of apoptotic thymocytes are immune-blotted with antibodies against CD63, TGS101, Alix, Hsp90 and β-actin, as indicated. Data were representative for two independent experiments. (b) EVs quantification: viable cells (n = 3) and apoptotic cells (n = 3) were cultured in serum-free medium for 6 hr, and EVs were extracted from the supernatant and quantified with BCA assay. Data were representative for two independent experiments. (c) TGFβ production of macrophages: peritoneal macrophages were cultured in serum-free medium (Med, n = 3) or stimulated with 0.5 × 106 apoptotic thymocytes (Thym, n = 3), EVs derived from 40 × 106 Thymocytes (Thym Exo, n = 3), or EVs derived from 20 × 106 Jurkat cell (Jurkat Exo, n = 3) for 24 hr. The total TGFβ levels in supernatants were quantified by ELISA. Data were representative for three independent experiments. (d) TGFβ mRNA in macrophages: peritoneal macrophages (n = 4) were stimulated with EVs for 0, 2, 4 and 8 hr. RNA was then extracted and quantified for TGFβ1 and GAPDH mRNA by qPCR. Data of three independent experiments were combined.
