Fig. 6: EV-induced activation of FAK is required for ITGB3-dependent EV uptake.

a Representative immunoblot analysis of pFAK activity either in normal conditions or clonogenic growth conditions with and without conditional medium (CM). b Internalization of 2–5 µg mL⁻¹ PKH26-labelled exosomes derived from MDA.MB.231 cells by MDA.MB.231 shCON, shITGB3, siFAK or shCON pretreated with 15 µM FAK 14 for 14–16 h, measured by FACS. c Representative immunoblot analysis confirming FAK knockdown and pFAK activity reduction after FAK-14 treatment. d Internalization of 2–5 µg mL⁻¹ PKH26-labelled exosomes derived from MDA.MB.231 cells by MDA.MB.231 shCON pCDNA3-GFP, shCON FAK-GFP, shITGB3 pCDNA3-GFP, and shITGB3 FAK-GFP, in GFP-positive cells measured by FACS. e Representative immunoblot analysis of pFAK activity in shCON pCDNA3-GFP, shCON FAK-GFP, shITGB3 pCDNA3-GFP, and shITGB3 FAK-GFP. f Representative immunoblot analysis of TSG101, CD81 and flotilin-1 on EVs derived from untreated MDA.MB.231 shCON or MDA.MB.231 shITGB3 cells or from control or FAK-14 inhibitor (15 μM) treated MDA.MB.231 shCON cells. g Representative immunoblot analysis of the autophosphorylation of FAK at Y397 as readout for its kinase activity. MDA.MB.231 shCON or shITGB3 serum-starved cells were pretreated with 20 μM Dyngo4a, 10 μM Pitstop 2 or 10 μg mL⁻¹ heparin for 30 min or with 15 μM FAK-14 for 14–16 h followed by addition of EVs for 60 min. h Model for the proposed role of ITGB3 in vesicle uptake and exosome biogenesis. (a) EV-HSPG interaction; (b) αvβ3 recruitment; (c) pFAK-DYNAMIN recruitment to endocytosis complex; (d) Dynamin-mediated internalization of EVs and EE formation. Source data are provided as a Source Data file. Model created with BioRender (https://biorender.com/). (**p value < 0.01, ***p value < 0.001 n = 3). Data are represented as mean ± SD in (b) and (d). Statistical analysis including two-way ANOVA multiple comparisons was carried out using GraphPad Prism 6.01.