Fig. 2

Effect of RD-LPS treatment viability and the function on EPCs. a RD-LPS protects against apoptosis of the lineage-negative stem cell population in the BM. BMCs were isolated, labeled with annexin V-FITC and To-Pro3, and measured by flow cytometry. b RD-LPS stimulates uptake of DiI-labeled ac-LDL by EPCs. Following 10 days of BMC culture in EBM-2 medium in the presence of vehicle PBS or 100 ng/ml RD-LPS, EPCs were incubated for 4 h with 1 μg protein/ml DiI-ac-LDL, as described in the “Materials and methods”. Cell-associated fluorescence was quantitated by flow cytometry. Each point represents the mean fluorescence of 10,000 cells. The experiment was repeated three times with similar results, n = 6, ***: p = 0.0002. Investigation of the role of sEVs in in vivo RD-LPS response. c RD-LPS-stimulated EPC response of the BM was abolished by in vivo GW4869 treatment (20 μg/mice), measured by flow cytometry (n = 5–6). One-way ANOVA, *p < 0.05 PBS vs. RD-LPS. Mean ± SD values are shown. d–f The characterization of sEVs isolated from the conditioned media of BMCs. d Representative transmission electron microphotographs (TEM) of sEVs isolated from the conditioned media of BMCs of PBS-injected (large image) and RD-LPS-injected mice (insert). e Particle size distribution and concentration in the sEV-enriched preparations isolated from the conditioned media of BMCs determined by tunable resistive pulse sensing (qNano). The upper and lower panels show representative profiles of sEVs in the PBS-injected and RD-LPS-injected groups, respectively. f Flow cytometry analysis of BMC-derived sEVs. Representative results of three independent experiments are shown. g Venn diagram of PBS control and RD-LPS-treated sEV proteome. The numbers of identified proteins are indicated. h Quantitative analysis of differentially expressed proteins in RD-LPS and PBS samples. Fold change threshold is > 4. Green- and pink-labeled proteins are down- and upregulated, respectively