fig2

Figure 2. NID1-enriched EVs facilitate DC maturation. (A) Flow cytometry analysis of changes in the expression of CD40, CD80, and MHC II on BMDCs after treatment with conditioned media from 1753^p53-/-cmyc with or without EVs; (B) Detection by Western blot of EV positive markers (Alix), negative markers (GM130, p62), and NID1 expression in the cell lysate and EVs collected from 1753^p53-/-cmyc cells; (C) NTA profiles of EVs isolated from 1753^p53-/-cmyc cells; (D) TEM imaging of EVs secreted by 1753^p53-/-cmyc cells. Scale bar: 100 nm; (E) Flow cytometry analysis showed changes in the expression of CD40 and MHC II on BMDCs after treatment with EVs derived from 1753^p53-/-cmyc; (F) Flow cytometry analysis of CCR7 expression on BMDCs after treatment with EVs derived from 1753^p53-/-cmyc cells; (G) ELISA determination of CCL17 levels in BMDC culture supernatants after treatment with EVs derived from1753^p53-/-cmyc cells at doses of 2 μg/mL and 10 μg/mL. Data are presented as the mean ± SD, with statistical significance determined by one-way ANOVA (multiple groups) (Tukey’s test) or Student’s t-test (two groups). Biological replicates are represented as individual data points. Western blot analysis was performed with three biological replicates. ^*P < 0.05; ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. NID1: Nidogen 1; EV: extracellular vesicle; DC: dendritic cell; BMDC: bone marrow-derived dendritic cell; CM: conditioned medium; MHC I: major histocompatibility complex class I; MHC II: major histocompatibility complex class II; CD40: cluster of differentiation 40; CD80: cluster of differentiation 80; CD11c: cluster of differentiation 11c; Alix: ALG-2-interacting protein X; NTA: nanoparticle tracking analysis; TEM: transmission electron microscopy; CCR7: C-C chemokine receptor 7; CCL17: C-C motif chemokine ligand 17; ELISA: enzyme-linked immunosorbent assay; SSC-A: side scatter-area; SD: standard deviation; ANOVA: analysis of variance; ns: not significant.