Fig. 4

BRCA1-KO fibroblasts plasma membrane and cancerous cells-derived exosomes are enriched for proteins involved in vesicles uptake. a Control [1] and BRCA1-KO [2] fibroblasts were lyzed and enriched for plasma membrane proteins as described under Materials and Methods. Isolated proteins were subjected to an SDS-PAGE to analyze isolation efficiency using an anti-β actin antibody (left panel) and the Plasma Membrane Fraction Western Blot Cocktail (right panel). Note that the plasma membrane-bound sodium/potassium (Na/K) ATPase and β-Actin were highly expressed, while the cytoplasmic GAPDH and nuclear Histone H3 are mildly or not expressed. b Protein samples were analyzed by mass spectrometry. We detected 1598 proteins. Gene Ontology (GO) analyses confirmed that the protein isolation from plasma membrane was efficient (1092 proteins are membrane-bound or proteins known to link to membrane proteins), with some contamination from other cell compartments. c The Venn diagram shows that there are 916 proteins shared, 169 expressed only in CTL and 513 expressed only in BRCA1-KO. d The number of proteins putatively involved in vesicles uptake. Note that BRCA1-KO fibroblasts express more proteins involved in vesicles uptake when compared to control fibroblasts. e-f BRCA1-KO and control fibroblasts were treated for 3 weeks with serum from patients with CRC-LM, or serum from healthy donor, respectively. Following 3 weeks of treatment, cells were maintained in exosome-free FBS to collect conditioned media. Exosomes were isolated from the respective conditioned media and subjected to mass spectrometry analyses. e Venn diagram: we detected 1265 proteins: 949 were shared proteins, 216 proteins were expressed only in CTL-exosomes and 100 proteins were expressed only in BRCA1-KO-exosomes. f The number of proteins putatively involved in vesicles uptake. Note that BRCA1-KO fibroblasts-derived exosomes express more ligands involved in vesicles uptake when compared to control fibroblasts-derived exosomes. g-h Graphs show the top list of proteins expressed in fibroblast plasma membrane (g) and exosomes (h), and that are involved in vesicles uptake. Data are mean +/− SD ((g) n = 3 plasma membrane preparations, and (H) n = 2 exosomes preparations. *P < 0.05, **P < 0.01, ***P < 0.001). Note that Y axis in H is in log scale