Figure 3. EVs derived from BMSCs are promoted by NSCs. The cells were grouped into Control (NSCs), Coculture (co-culture of BMSCs and NSCs), Coculture-DMSO (co-culture of BMSCs and NSCs with DMSO in the apical chamber), Coculture-GW4869 (co-culture of BMSCs and NSCs cells with GW4869 in the apical chamber). (A) Co-culture of BMSCs and NSCs in the Transwell chambers. (B) The viability of neuronal cells after GW4869 (an inhibitor for EVs) treatment determined using CCK-8 assay. (C) The mRNA expression of neuronal cell markers (MAP-2 and Tuj-1) in the BMSCs untreated or co-cultured with BMSCs with the addition of DMSO or GW4869 in the apical chamber determined by RT-qPCR. (D) The concentration of EVs determined by NTA. (E) The expression profile of miR-133b in BMSCs-derived EVs determined by RT-qPCR. (F) The neuronal cell apoptosis assessed by TUNEL assay (× 200). (G) The neuronal cell proliferation after co-culture with EVs detected by CCK-8 assay. * p < 0.05 vs. NSCs or BMSCs alone. # p < 0.05 vs. co-culture of BMSCs and NSCs. Data are expressed as mean ± standard deviation, and data between two groups were analyzed using the unpaired t test while data among multiple groups were analyzed by one-way ANOVA with the Tukey's post hoc test. The cell experiments were conducted 3 times independently.