Fig. 2

sEVs hyperactivate the TGF-β signaling activity in breast cancer cells in vitro. a TGF-β/SMAD3 signaling reporter (Ad-CAGA-Fluc) activity quantified in breast cell lines treated ±5 ng/mL rhTGF-β1 for 24 h. b sEV secretion was quantified by BCA assay. c TGF-β activity in sEVs secreted by breast cell lines was quantified and normalized per µg of sEV total protein. TGF-β/SMAD3 signaling reporter (Ad-CAGA-Fluc) activity quantified in (d) MDA231 and (f) MCF7 cells treated with rhTGF-β1 or MDA231-sEVs (increasing concentrations). Results were normalized by Gaussia luciferase (Ad-CMV-Gluc) activity. Phosphorylated (p)SMAD2 levels analyzed in (e) MDA231 and (g) MCF7 cells treated with rhTGF-β1 or MDA231-sEVs (1 h; increasing concentrations). Total (t)SMAD2 and β-actin were used as loading controls. h–k TGF-β/SMAD3 signaling activity was quantified in cancer cells as in (a). h MDA231 and (i) MCF7 cells infected with Ad-CMV-Flag-SMAD7. Ad-CMV-GFP: control adenovirus. j MDA231 and (k) MCF7 cells treated with SB431542 ± rhTGF-β1 or MDA231-sEVs. DMSO: vehicle for SB431452. pSMAD2 levels were evaluated in (l) MDA231 and (m) MCF7 cells treated with rhTGF-β1 or MDA231-sEVs ± SB431542 as in (e, g). DMSO: vehicle for SB431542. Results represent mean ± SD (n ≥ 3). One-way ANOVA test followed by Dunn’s Multiple Comparison test were used to analyze data in (a–c, h–k). Unpaired Student’s t-test was used to analyze data in (d, f). ns: statistically non-significant, *p < 0.05, **p < 0.01, ***p < 0.001