Figure 3
Extracellular and intracellular distributions of vesicle-loaded tRNAGlu under oxidative stress. BUMPT cells were treated with the indicated concentrations of H2O2 for 1 h and thereafter conditioned for 23 h with serum-free, H2O2-free growth media. Note that cell viability was not significantly affected in this condition. (a) Immunoblotting analysis showing the indicated proteins in extracellular vesicle (EV) fraction, validating the extracellular vesicle isolation protocol used in this study. RCAS1 was used as negative control. (b) Relative tRNAGlu abundance in extracellular vesicles from cells under oxidative stress. Benzonase was used to degrade RNAs associated nonspecifically with the surface of extracellular vesicles. *p < 0.05. (c) Total number of extracellular vesicles (EV) from cells under oxidative stress. Note that transient oxidative stress decreases release of extracellular vesicles. *p < 0.05. (d) Relative abundances of intracellular vesicle tRNAGlu, tRNAAspGTC, and tRNAValAAC under oxidative stress. Benzonase was used to degrade cytoplasmic RNAs associated nonspecifically with the surface of intracellular vesicles. Note that transient oxidative stress resulted in reduced loading of tRNAGlu and tRNAAspGTC in intracellular vesicles. *p < 0.05. (e) Transport of tRNAGlu to SDCBP or TSG101-positive endosomes under oxidative stress. A 0.4–1.8 M continuous sucrose gradient was used to separate intracellular vesicles enriched with exosome cargo proteins, SDCBP and TSG 101. Post-nuclear supernatants were pelleted at 100 K × g, and the resuspended pellets were top-loaded on the gradient to separate intracellular vesicles, based on their size and density. Note that benzonase was used to degrade cytoplasmic RNAs. Total RNA measurement, RT-qPCR, and western analysis were performed as described in “Methods” section. *p < 0.05.
