Fig 1: Rapamycin-induced loading of FKBP12-mCherry-Cargo into extracellular vesicles (EVs) containing CD9-EGFP-FRB. All data were analyzed via one-way ANOVA, followed by Turkey’s HSD test. (A) COS-7 cells were transiently transfected with the expression plasmids encoding CD9-EGFP-FRB and mCherry-FKBP12-Cargo. The cells were cultured in complete media for 24 h and then incubated with indicated rapamycin under the serum starvation condition. The expression levels of EGFP and mCherry were confirmed in cell lysates by immunoblot analysis. Actin was used as a loading control. (B) Quantification of panel (A). The quantification was performed with ImageJ. The fold change (ratio of mCherry/EGFP) was calculated between pairs of plasmid expression levels from three independent immunoblot analyses. n = 3, mean ± standard deviation (SD) (ns = not significant). (C) The EVs were isolated according to materials and methods described in the detailed isolation methods of EVs. Several proteins such as Alix, CD81, and HSP70 were enriched in EVs and used as markers. (D) Quantification of panel (C). n = 4, mean ± standard deviation (SD). There are significant differences between the absence or presence of rapamycin treatment groups. (ns = not significant, ** p = 0.0031 for rapamycin) (E) The transfected COS-7 cells were cultured in complete media for 24 h and then incubated with 100 nM rapamycin for different times under the serum starvation condition. The EVs were isolated according to materials and methods. (F) Quantification of panel (E). n = 3, mean ± standard deviation (SD). There are significant differences between 0 h and 24 h in 100 nM rapamycin treatment groups. ** p = 0.0047 for 24 h compared to 0 h. All raw data from immunoblots are in Supplementary Figure S6.