Figure 3
The mechanisms of TGFβ production in macrophages stimulated with EVs. (a) Annexin V inhibits the TGFβ production in macrophages stimulated by EVs: macrophages were pretreated with Annexin V (n = 5) or medium (n = 5) for 1 hr, then stimulated with or without EVs for 24 hr. The TGFβ was quantified in supernatant by ELISA. Data of five independent experiments were combined. (b) PI3K, AP-1 and p38 MAPK pathways are not involved: macrophages were pretreated with PI3K (BYL719 1 µM, BKM120 1 µM), Akt (MK-2206 1 µM), AP-1 (SR11302 1 µM) and p38 MAPK (SB203580 10 µM) inhibitors for 1 hr, then stimulated with (MΦ + EVs, n = 3) or without EVs (MΦ, n = 3) for 24 hr. The TGFβ was quantified in supernatant by ELISA. Data were representative for two independent experiments. (c) The expression of FOXO3 and pFOXO3 in macrophages stimulated with EVs: macrophages were treated with EVs for 0, 2, 4, 6 and 8 hr, then harvested and immunoblotted with antibodies against FOXO3, pFOXO3 and αTubulin. Data were representative for two independent experiments. (d) FOXO3 Knockdown in macrophages: macrophages were transfected with FOXO3 siRNA (n = 4) or control siRNA (n = 4). The mRNA and protein level of knockdown efficiency was confirmed at 24 and 48 hr. Data of four independent experiments were combined. (e) FOXO3 is implicated in the mechanism: macrophages were transfected with FOXO3 siRNA (n = 4) or control siRNA (n = 4) for 24 hr, then treated with EVs for 24 hr. The TGFβ was quantified in supernatant by ELISA. Data of two independent experiments were combined. (f) Scheme of EV-driven TGFβ pathway in macrophages.
