Fig. 3
Construction and characterization of EVs-siBRAFV600E. (A) BRAF mRNA levels were analyzed by qRT-PCR. BRAF mRNA levels were measured after treating HEK293T, COLO320, and RKO cells with 50 µg/mL of EVs-siBRAFV600E for 24 h. (B) Construction of the therapeutic agent. (C) NTA analysis of EVs-NC (blue) and EVs-siBRAFV600E (red). (D)(E) TEM images of eVs-NC (D) and EVs-siBRAFV600E (E); scale bar: 200 nm. (F) WB analysis of the extracellular vesicle marker proteins, TSG101, CD81 and CD63. (G) Fluorescence microscopy images showing the uptake of EVs-NC and EVs-siBRAFV600E by RKO cells. The cell nuclei were stained with DAPI, and the EVs were stained with DiD; scale bar: 20 μm. (H) qRT-PCR analysis of siBRAFV600E levels in EVs-NC and EVs-siBRAFV600E. (I) WB analysis of BRAF protein levels. After COLO320 and RKO cells were treated with PBS, or 50 µg/mL of EVs-NC or EVs-siBRAFV600E for 24 h, cell proteins were extracted to measure BRAF protein expression. The data are reported as the means ± SDs of the experiments (n = 3). Two-tailed Student’s t-tests for (a) and (h), and one-way ANOVA followed by Tukey test multiple comparisons for (I), *p < 0.05, **p < 0.01 and ***p < 0.001