Fig 2: EVs from the RBC concentrates of healthy donors display heterogenous diameters and are LPS-positive. (A) Image extracted from a nanoparticle tracking analysis (NTA) video. EVs were counted and their diameter was measured based on their Brownian motion. (B) The table shows an example of the results obtained with the sample further reported as being analyzed by flow cytometry. The mean and mode of five video acquisitions performed for the NTA measurements are shown ±SD. (C) Histogram of the distribution of the diameter of the EVs. (D) Immunoblot detection of Alix, Actin, CD63, and LPS in the RBC EVs. Experiments conducted with the EVs from blood donor EV#6 (see Table 1).

Fig 3: MSC promotes AML cell differentiation by EVs. (A) Electron microscopy image of EVs isolated from the conditional medium of MSC. Scale bar = 200 nm. (B) Western blotting for the expression of Alix, Flotillin-1, Annexin V and NE in MSC, supernatant and EVs which were extracted from MSC conditional medium, respectively. Data are quantified on the right. (C) Confocal microscopy images of U937 cells treated with EVs derived from CFSE-labelled MSC for 2 h. 100 × magnification; scale bar = 20 μm; (D) U937 cells were cultured with EVs (10, 20, and 30 μg) for 48 h. The myeloid differentiation markers CD11b and CD14 in U937 cells were analyzed by flow cytometry (n = 3). (E) U937 cells were cultured with MSC in the presence of EVs trafficking inhibitor Y27632 (10, 20, and 40 μmol/L) for 48 h. The myeloid differentiation markers CD11b and CD14 in U937 cells were analyzed by flow cytometry (n = 3). (F) MSC EVs (30 μg) were added to stimulate U937 cells. Western blotting was applied to detect the levels of phosphorylation of p38 and STAT3 in U937 cells. Data are quantified on the right. (G) U937 cells were treated by the p38 MAPK inhibitor SB239063 (SB) (10 μmol/L) and/or the STAT3 inhibitor Stattic (5 μmol/L) followed by incubating with MSC for 48 h. The percentage of CD14+CD11b+ cells was determined by flow cytometry. (H, I) Flow cytometry analysis of the percentage of leukemic cells (CD45 positive cells) in peripheral blood (PB) (I) and bone marrow (BM) (J) in vehicle, MSC-treated and MSC-EVs-treated mice after 25 days of transplantation (n = 4 or 5 each group). (J, K) Flow cytometry analysis of the expression of the differentiation marker CD14 in PB (K) and BM (L) derived leukemic cells (n = 4 or 5 each group). (L) Fluorescent images of the AML cells infiltration in the spleen as observed via immunofluorescence with anti-CD45 antibody (green). Nuclei are stained with DAPI (blue). 10 × magnification; scale bar = 100 μm. Data are expressed as the mean ± SEM. Data were analyzed by Student's t test (E, F) and one-way ANOVA followed by Tukey's test (D, G–K). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig 4: VDR modulates the production and release of NE carried EVs. (A) Luciferase reporter assay evaluating the transcriptional activity of VDR, following treatment with 1,25D3 (n = 6). (B) Relative NE protein level in DMSO- or 1,25D3-treated MSC was evaluated by Western blotting (n = 3). (C) Confocal microscopy analysis of the NE expression (Alexa Fluor 488) in DMSO- or 1,25D3-treated MSC. Data are quantified on the right. 100× magnification; scale bar = 20 μm. (D) The NE expressions in MSC & shcon or MSC & shVDR treated by U937 cells with or without 1,25D3 were assessed by RT-qPCR (n = 3). (E) The NE expression in MSC OE-con or MSC OE-VDR treated by U937 cells with or without 1,25D3 was assessed by RT-qPCR (n = 3). (F) MSC was cultured in the presence or absence of 1,25D3. The expressions of Alix, Flotillin-1, NE in MSC-EVs were detected by Western blotting (n = 3). Data are quantified on the right. (G) EVs isolated from 1,25D3-primed MSC (pre-EVs) were added to treat U937 cells. The myeloid differentiation markers CD11b and CD14 in U937 cells were analyzed by flow cytometry. (n = 3) (H) The numbers of EVs existed in MSC & shcon or MSC & shVDR medium were assessed by flow cytometry (n = 3). (I) U937 cells were treated by MSC or MSC VDR-KO in the presence with or without 1,25D3. The expressions of CD14 and CD11b in U937 cells were evaluated by flow cytometry (n = 3). All statistical data in this figure are expressed as the mean ± SEM. Data were analyzed by Student's t test (B, C), two-way ANOVA followed by Bonferroni's test (D, E, F, I) and one-way ANOVA followed by Tukey's test (B, C, H). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig 5: Synaptic NMDAR activation triggers secretion of EVs containing Notch1 and Notch2 proteins(A) Experimental scheme depicting EV purification workflow and cargo analyses.(B) Representative images of EV particles present in SEC fraction 2 (F2) visualized by negative staining EM. Scale bar, 500 nm; inset scale bar, 50 nm.(C) The diameter range of EV-like particles. n = 299 particles from two biological replicates.(D) The EV markers Alix, Tsg101, and CD81 but not GM130 are selectively detected in SEC F2 isolated from glycine-stimulated neuronal cultures.(E) Proteomic analysis reveals that Notch1 and Notch2 are highly abundant in SEC F2 along with other known EV protein markers. Mean ± SEM from 3 biological replicates. Insert: Notch1 and Notch2 peptides identified by MS/MS.(F) WB validation of Notch1ICD and Notch2ICD in SEC F2 (top). Silver-stained gel indicating the total amount of protein loaded across the 10 fractions (bottom). See also Figure S1 and Table S1.