Fig 1: Proteomic analysis found that LRG1 enriched in platelet-derived exosomes.A Proteomic analysis of platelet-rich plasma derived from healthy individuals or MM patients, and a heat map showing the differential proteins. B GO analysis was performed on the differential proteins, and cell component analysis found that the differential proteins were closely related to vesicle secretion. C GSEA analysis of differential proteins found that differential proteins were closely related to EMT and angiogenesis. D Volcano plot showing significantly different proteins found in proteome analysis. E The expression of S100A8, S100A9, MPO, SAA1, and LRG1 in platelet-rich plasma derived from healthy people or patients with MM was verified by WB, and the expression of LRG1 was significantly increased in platelet-rich plasma derived from patients with MM. F The exosomes in platelet-rich plasma derived from MM patients were obtained, and the morphology and particle size of exosomes were analyzed by transmission electron microscopy and nanoparticle tracking analysis (NTA). G Exosomes expressing the specific platelet marker CD41 and exosome marker CD63 were sorted by flow cytometry. H The expression of LRG1 in platelet-derived exosomes was verified by WB. Compared with the control group, LRG1 expression was significantly increased in MM platelet-derived exosomes. ***P < 0.001.
Fig 2: Interaction between LRG1 and OLFM4.A, B Proteomic analysis of possible interacting molecules of LRG1, of which OLFM4 is a predicted interacting molecule of LRG1 and is closely related to VEGF and N-cadherin. C Co-IP detection of interactions between LRG1 and OLFM family molecules. LRG1 can interact with OLFM4. D TGF-β enhances the interaction between LRG1 and OLFM4. E Different truncated LRG1 expression plasmids were constructed. Co-IP found that LRG1 without LRRs domain could not interact with OLFM4. E Different truncated OLFM4 expression plasmids were constructed. Co-IP found that OLFM4 without the Olfactomedin domain could not interact with LRG1.
Fig 3: LRG1 inhibitor reversed the pro-proliferation effect of MM platelet-derived exosomes on U266 and RPMI8226 cells.A PKH26 was used to label exosomes, and wheat germ agglutinin (WGA, green) was used to label the membrane. The fusion of exosomes with U266 cells was observed under a fluorescence microscope. DAPI stained the nuclei. B Edu assay to detect cell proliferation. Compared with the MM platelet-derived exosome plus IgG group, the LRG1 inhibitor significantly reduced the number of Edu-positive cells in U266 and RPMI8226 cells. C Compared with the MM platelet-derived exosome plus IgG group, the LRG1 inhibitor significantly increased the number of apoptotic cells in U266 and RPMI8226. D, E LRG1 inhibitor significantly increased the expression of Bax and decreased the expression of Bcl2 compared with the MM platelet-derived exosome plus IgG group. *P < 0.05.
Fig 4: High LRG1 expression is a poor prognostic factor in MM patients.Survival curve analysis of the relationship between the expression of platelet-derived exosomal LRG1 and the overall survival and disease-free survival of MM patients. MM patients with high LRG1 expression had shorter overall survival and disease-free survival.
Fig 5: LRG1 promotes EMT and angiogenesis through OLFM4 in vitro.A Representative immunofluorescence images of E-cadherin, N-cadherin, Vimentin, VEGF. LRG1 significantly inhibited the expression of E-cadherin and increased the expression of N-cadherin, Vimentin, and VEGF, while OLFM4 siRNA transfection significantly reversed the effect of LRG1 on these molecules. B The expression of E-cadherin, N-cadherin, Vimentin, and VEGF was detected by WB. C In vitro blood vessel formation assay. LRG1 significantly increased branch points and capillary length in vascular endothelial cells in vitro, whereas OLFM4 siRNA transfection significantly attenuated the effect of LRG1 on angiogenesis. *P < 0.05, **P < 0.01.
Supplier Page from Abcam for Human LRG ELISA Kit (LRG1)