Fig. 3 | Signal Transduction and Targeted Therapy

Fig. 3

From: Simultaneously targeting extracellular vesicle trafficking and TGF-β receptor kinase activity blocks signaling hyperactivation and metastasis

Fig. 3

Rab27a knockdown decreases sEV secretion and inhibits the TGF-β signaling activity in breast cancer cells in vitro. sEVs were isolated from parental (P) MDA231 and MDA.Rab27a.shRNA (RAB27A knockdown (KD)) cell culture conditioned medium. a sEV secretion was quantified by BCA assay. b, c Particle size distribution and concentration evaluated by NTA. d Alix and TSG101 expression assessed by western blot in (P) and (KD) whole cell lysates (WCL) and sEVs. e, f pSMAD2 levels evaluated in (P) and (KD) cells treated (e) with 2 ng/mL rhTGF-β1 (increasing time intervals) or (f) with indicated rhTGF-β1 concentrations (8 h). Total (t)SMAD2 and β-Actin were used as loading controls. g, h The TGF-β/SMAD3 signaling reporter (Ad-CAGA-Fluc) activity was quantified in parental and RAB27A KD cells treated ± rhTGF-β1 for (g) 24 h or (h) 48 h. Results normalized by Gaussia luciferase (Ad-CMV-Gluc) activity. Results represent mean ± SD (n ≥ 3). Unpaired Student’s t-test used for comparison. **p < 0.01, ***p < 0.001

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