Fig. 4

TGF-β signaling is inhibited by DMA, Heparin and PNP-Xyl in vitro. a–d sEVs were isolated from MDA231 cell culture conditioned medium after treatment ± 100 µM DMA (2 h). a sEV secretion quantified by BCA assay. b, c Particle size distribution and concentration evaluated by NTA. d pSMAD2 levels were assessed by western blot in MDA231 cells treated ± DMA (100 µM) and rhTGF-β1 (5 ng/mL). Total (t)SMAD2 and β-actin were used as loading controls. e, f TGF-β/SMAD3 signaling reporter (Ad-CAGA-Fluc) activity quantified in (e) MDA231 and (g) MCF7 cells treated ± rhTGF-β1 (5 ng/mL) ± DMA (24 h). Luciferase assay results normalized by Gaussia luciferase (Ad-CMV-Gluc) activity. g pSMAD2 levels assessed in MDA231 cells treated with rhTGF-β1 or MDA231-sEVs after treatment ± heparin. h, i TGF-β/SMAD3 signaling activity quantified in (h) MDA231 and (i) MCF7 cells treated with MDA231-sEVs ± heparin as in (e, f). j pSMAD2 levels assessed in MDA231 cells treated with rhTGF-β1 or MDA231-sEVs after treatment ± PNP-Xyl as in (d, g). k, l TGF-β/SMAD3 signaling activity quantified in (k) MDA231 and (l) MCF7 cells treated with MDA231-sEVs after treatment ± PNP-Xyl (24 h). Results represent mean ± SD (n ≥ 3). Unpaired Student’s t-test was used to analyze data in (a, c). One-way ANOVA test followed by Dunn’s Multiple Comparison test was used to analyze data in (e–f, h–i, k–l). ns: statistically non-significant, **p < 0.01, ***p < 0.001