Fig 2: Neuropilin-1 expression emerges as a co-dependent factor of integrins in GBM. (A–F) The Cancer Genome Atlas (TCGA RNAseq dataset) was interrogated via the GlioVis platform to analyze differential gene expression in 528 GBM patients. Patients were clustered between low or high ITGB1 mRNA expression (median cut-off). Representation of heatmap (A) and Pearson’s correlation factors (B) between each differentially expressed gene and ITGB1, with a focus on the ones with a positive correlation ranging from 0.7 to 0.8. Pearson’s correlation heatmap analysis of the association between NRP1 and NRP2 mRNA expression level and each ITGB subunit (B1 to B8) (C). NRP1 mRNA expression from non-tumor samples, as compared to GBM patient samples (D). Kaplan-Meier survival curve (percent) for 528 GBM patients clustered between low or high NRP1 mRNA level, using median cut-off (E). Down and upregulated genes associated with NRP1 were analyzed for their GO (gene ontology) functions. Each GO function is shown with its corresponding p-value (-log10) (F). (G–I) Glioblastoma stem-like patient-derived cells (GSC1 and GSC9) were transfected with non-silencing (sic, black) and NRP1 targeting duplexes (si NRP1 , blue). The knockdown efficiency and the level of ITGB subunits (B1 to B8) was analyzed by RT-qPCR (G) and western-blot (H). ACTB and HRPT1 were used as housekeeping genes for normalization in (G) and GAPDH was used as a loading control (H). nd, not detected. Cell surface flow cytometry analysis in GSC1 and GSC9 using the indicated antibodies for ITGB1 and ITGB3. Histograms present the mean fluorescence intensity (MFI) normalized to sic. Data are representative of three different experiments (I). (J) CRISPR-Cas9 gene editing-mediated depletion of NRP1 was evaluated in NRP1 KO GSC1 (light blue) at the RNA and protein level, and compared to WT GSC1 (dark grey). qPCR analysis of NRP1 mRNA is shown as the mean ± s.e.m. fold change from three independent experiments using ACTB and HRPT1, as housekeeping genes for normalization (left panel). Representative plots of flow cytometry analysis show the staining of immunoglobulin isotype control (light grey), WT (dark grey), and NRP1 KO (light blue). (K) The level of ITGB3 was analyzed by RT-qPCR (left panel) and by cell surface flow cytometry (right panel). ACTB and HRPT1 were used as housekeeping genes for normalization. All panels are representative of at least three independent experiments, t-test and ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: Characterization of platelet-derived extracellular vesicles (PEVs) in sepsis patients and healthy volunteers. A: The morphology of extracellular vesicles (EVs) isolated from clinical blood samples was observed by transmission electron microscopy (TEM); B: The diameter of EVs isolated from clinical blood samples was analyzed by nanoparticle tracking analysis (NTA); C: The proportion of EVs with surface markers CD41 + or CD61 + in isolated EVs was detected by flow cytometry. D: Comparison of the number of PEVs (CD41 + EVs) in clinical blood samples from normal healthy volunteers and patients with sepsis, **P < 0.01 vs. Normal group. Normal (n = 30) and Sepsis (n = 30) group, EV: extracellular vesicle; TEM, transmission electron microscopy; NTA, nanoparticle tracking analysis

Fig 4: Plasma EV test sample (PEVTES) stability during storage. Concentration of platelet (CD61+, A–C), erythrocyte (CD235a+, D–F) or lactadherin+ (G–I), immuno-fluorescently stained EVs stored in dimethyl sulfoxide (DMSO; 10% f.c.), glycerol (20% f.c.) or trehalose (0.5 M f.c.) at -80 °C for 12 months. Samples were measured fresh (red triangle), after freeze thawing after 1, 3, 6 and 12 (black circle) months of storage with a calibrated flow cytometer (Apogee A60-Micro, Apogee Flow Systems). Data are presented as mean ± standard deviation EV concentration (mL-1) versus the storage time (months). For details, please see Supplementary Material S1.

Fig 5: Characterization of PEVs in rat plasma. A: TEM was utilized to observe the morphology of PEVs in rat blood; B: NTA was employed to analyze the diameter of PEVs in rat blood; C: Flow cytometry was applied to detect the proportion of EVs and CD41- and CD61-positive PEVs. PEV, platelet-derived extracellular vesicles; TEM, transmission electron microscopy; NTA, nanoparticle tracking analysis