Translocation of KRS from the MSC to exosomes. (A) Dissociation of KRS and EPRS from the MSC was monitored by gel filtration of proteins that were extracted from HCT116 cells incubated in normal (control; Con) and starvation (Starv) conditions. EPRS is a known component of MSC (Quevillon et al., 1999). (B) The effect of caspase-8 inhibitor (Z-VAD-ETD) on the starvation-induced dissociation of KRS from MSC in HCT116 cells was determined by coimmunoprecipitation of KRS and EPRS. The proportion of KRS dissociated from MSC was determined by immunoblotting the supernatant (ID) after removing the coimmunoprecipitates of KRS and EPRS (IP). The cellular levels of KRS and EPRS were determined in whole-cell lysates (WCLs). 100% and 15% of IP and ID samples were loaded for gel electrophoresis, respectively. (C) The effects of ΔN12 and D12A mutations and syntenin (syn) knockdown on the dissociation of KRS-Myc from MSC were determined as in B. (D) The effects of ΔC5 (the C-terminal 5-aa deletion) on the dissociation of KRS-Myc from the MSC were determined as in B. (E) The effect of caspase-8 inhibitor on the association and colocalization of KRS-syntenin-Alix was shown by BiFC and fluorescence microscopy, respectively. The starvation-induced association of KRS and syntenin was shown by BiFC (green) as described in Fig. 2 B. The localization of KRS–syntenin complexes revealed by BiFC was further analyzed with mCherry-Alix (red). Nuclei were visualized by DAPI staining (blue). (F) Immunogold-staining EM analysis showing the localization of KRS in vesicles located within the MVB of HCT116 cells under starvation conditions. Arrowheads in each panel indicate the localization of immunogold-labeled KRS. Bars: (F1, F3, and F4) 200 nm; (F2) 100 nm.