fig3

Figure 3. EV-NID1 promotes differentiation of DCs into mregDCs. (A) Western blot demonstration of NID1 expression in 1753^p53-/-cmyc NID1 NC and NID1 KD cells; (B) Flow cytometric analysis of CD86 expression in BMDCs following co-culture with 1753^p53-/-cmyc NID1 NC and NID1 KD cells; (C) Detection by Western blot of EV positive marker (Alix), negative marker (GM130), and NID1 expression in cell lysate and EVs of 1753^p53-/-cmyc NID1 NC and NID1 KD cells; (D) NTA profiles of EVs isolated from 1753^p53-/-cmyc NID1 NC (left) and NID1 KD (right) cells; (E) TEM imaging of EVs secreted by 1753^p53-/-cmyc NID1 NC (left) and NID1 KD (right) cells. Scale bar: 100 nm; (F) Flow cytometry analysis of changes in the expression of CD80 and CD86 on BMDCs after treatment with NID1 NC and NID1 KD EVs; (G) Flow cytometry analysis of changes in the expression of PD-L2, TIM-3 and CD200 after treatment with NID1 NC and NID1 KD EVs; (H) ELISA determination of CCL17 levels in BMDC culture supernatants after treatment with NID1 NC or KD EVs (10 μg/mL). Data are presented as the mean ± SD, with statistical significance determined by one-way ANOVA (Tukey’s test). 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; NC: negative control; KD: knockdown; EV: extracellular vesicle; DC: dendritic cell; mregDC: mature regulatory dendritic cell; BMDC: bone marrow-derived dendritic cell; CD80: cluster of differentiation 80; CD86: cluster of differentiation 86; CD200: cluster of differentiation 200; Alix: ALG-2-interacting protein X; NTA: nanoparticle tracking analysis; TEM: transmission electron microscopy; PD-L2: programmed death-ligand 2; TIM-3: T-cell immunoglobulin and mucin-domain containing-3; LPS: lipopolysaccharide; CCL17: C-C motif chemokine ligand 17; ELISA: enzyme-linked immunosorbent assay; SD: standard deviation; ANOVA: analysis of variance; ns: not significant.