Fig 1: Isolation and identification of plasma exosomes from gastric cancer patients. (a) Transmission electron microscopy for observing the morphological characteristics of isolated exosomes. Bar = 100 nm. (b) Western blot for detecting the expression of exosome markers CD63, CD9, TSG1010, and GM130. (c) Nanoparticle tracking analysis for detecting the particle size and concentration of exosomes.
Fig 2: ER stress up-regulated breast cancer cell-derived exosomal miR-27a-3p expression. A, GRP78, PERK, ATF6 and IRE1a mRNA expression in breast cancer cells was determined using RT-qPCR, *P < 0.05 vs normal cells. B, GRP78, PERK, ATF6 and IRE1a protein expression in breast cancer cells was determined using Western blot assay, *, vs normal tissues, P < 0.05. C, Transmission electron microscope was used to evaluate exosomes (scale bar, 100 nm). D, Diameter distribution detected using Image-Pro Plus software. E, Flow cytometry was adopted to measure the content of exosome surface marker CD63. F, Contents of CD63, CD9, CD81, Tsg101 and GRP94 were observed using Western blot assay. G, miR-27a-3p expression was determined in MCF-7 and MDA-MB-231 breast cancer cells, *, vs control (MCF-7), P < 0.05; #, vs control (MDA-MB-231), P < 0.05. H, miR-27a-3p expression in MCF-7 and MDA-MB-231 breast cancer cell-derived exosomes was assessed using RT-qPCR, *P < 0.05, vs control. I, miR-27a-3p expression in tumour tissues and paracancerous tissues. J, correlation analysis of miR-27a-3p expression and survival of breast cancer patients. Measurement data were presented as mean ± standard deviation. The unpaired t test was used to analyse the differences between two experimental groups, and ANOVA was utilized to analyse data among multiple groups, followed by Tukey's post hoc test
Fig 3: Characterization of HASC-proliferating EVs (P-EV), HASC-white adipogenic differentiating EVs (D-EV), and HASC-beige adipogenic differentiating EVs (BD-EV).(A) TEM and (B) cryo-TEM images of P-EV, D-EV, and BD-EV. (C) Immunoblotting for CD9, CD63, ALIX, and TSG101 of P-EV, D-EV, and BD-EV. (D) The size distributions of P-EV, D-EV, and BD-EV were analyzed by dynamic light scattering. (E) Confocal laser scanning microscopic images of HASCs after the 3-hour incubation with 1 × 108 particles of DiD-labeled D-EV and PKH26-labeled P-EV and BD-EV. The nontreated group was used as control. Scale bar, 20 µm. (F) WST-1–based colorimetric assay to quantify proliferation and viability of HASCs after 1 and 3 days of culture with D-EV and BD-EV (*P < 0.001). Data are shown as the means ± SD from three separate experiments. GM, growth medium; BDM, beige adipogenic differentitation medium.
Fig 4: Identification of human SMSCs and extracellular vesicles. a SMSCs exhibited a spindle-like morphology. b SMSCs showed multidirectional differentiation potential of osteogenesis, adipogenesis, and chondrogenesis. c Characteristic cell surface markers of SMSCs were analyzed by flow cytometry. d EVs and LPS-pre EVs observed through TEM. e Particle size distribution of EVs and LPS-pre EVs analyzed by NanoSight. f extracellular vesicles markers (CD9, CD63, Alix, TSG101) analyzed by western blot. g Chondrocyte internalization of EVs and LPS-pre EVs as observed under fluorescence microscope
Fig 5: GW4869 administration inhibits exosome release. Seven days post either DMSO or GW4869 administration, exosomes were collected from ischemic and sham brain extracellular spaces for NTA (A) and western blot (B) analyses. (A) The concentration of exosome suspension was determined by NTA. (B) Representative immunoblots of CD9 and Flot1 were shown along with quantifications of protein expression levels. Western blot data were presented as fold change compared with those in sham rat brains. Error bars denote s.d. from triplicate measurements. *p < 0.05 by two-tailed t-test (n = 9). #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. the ischemia with DMSO treatment group by two-tailed t-test (n = 9).
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