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. 2019 Apr 13;9(8):2346–2360. doi: 10.7150/thno.29945

Figure 2.

Figure 2

MiR21-loaded EVs are functional in vitro. (A) Schematic representation of pCMV-luc-PDCD4 3'untranslated regions (UTR), the PDCD4 3'UTR reporter vector (upper panel). CMV, cytomegalovirus promoter. FLuc, firefly luciferase. HEK293T cells were co-transfected with pCMV-luc-PDCD4 3'UTR and pRL-TK Renilla luciferase plasmid. Four hours after transfection, the cells were treated with Ctrl-EVs or various doses of miR21-EVs, as indicated. After 24 h, firefly luciferase activity was determined and normalized to Renilla luciferase activity (lower panel). Data shown as mean ± SEM. **, p < 0.01 compared with Ctrl-EVs group. (B) Confocal microscopy images of miR21-EVs uptaking by H9c2 cells, HUVECs, and primary cultured neonatal mouse cardiomyocytes (CMs), respectively. MiR21-EVs were labeled with red fluorescent dye PKH26 and incubated with indicated cells for 4 h. The regions boxed with white dashed lines were enlarged on the right. PKH26-labeled miR21-EVs were indicated by yellow arrowheads. Scale bar, 50 μm. (C) Quantification of miR21-EVs positive/negative cells of the corresponding images of (B) (n = 8 per cell line). (D) Analysis of mature miR21 levels by qPCR in H9c2 cells, HUVECs and CMs that were incubated with PBS, Ctrl-EVs or mi21-EVs, respectively, for 12 h. For comparison, miR21 level in PBS group was set as 1. Data shown as mean ± SEM. **, p < 0.01 compared with PBS group. (E) H9c2 cells, HUVECs, and CMs were treated with or without H2O2 and simultaneously incubated with PBS, Ctrl-EVs, or miR21-EVs, respectively. (F) H9c2 cells and HUVECs were treated with Ctrl-antagomir or miR21-antagomir (200 nM) in the presence of H2O2 and simultaneously incubated with Ctrl-EVs or miR21-EVs, respectively. After 12 h, PDCD4 protein level in (E and F) was determined by Western blot analysis. The working concentration of H2O2 was 400 μM in HUVECs and 100 μM in H9c2 cells and CMs, respectively. The ratio of PDCD4 to β-actin or GAPDH (relative band intensity) was measured using the Image-Pro Plus software. Data shown as mean ± SEM. *, p < 0.05, **, p < 0.01, N.S., no significant. The work concentrations of Ctrl-EVs and miR21-EVs were 2 μg/mL in (B-F).