Skip to main content
. 2018 Aug 6;217(8):2891–2910. doi: 10.1083/jcb.201710170

Figure 6.

Figure 6.

EVs contain, release, and synthesize cAMP. (A) Bar graph of cAMP TR-FRET data showing cAMP levels from lysed EVs derived from either ACA-YFP/aca−, WT, or aca− cells. 0.5 µg of purified preparation obtained from 16.8 × 108 vesicles was used in the assay. Results presented as mean ± SEM of three independent experiments. (B and C) Time-dependent release of cAMP from intact (B) and lysed (C) EVs derived from ACA-YFP/aca−, WT, or aca− cells. cAMP measurements were made every 15 min for 90 min after a 60-min equilibration incubation. Insets: Illustration depicting the principles of the cAMP TR-FRET assay. Results presented as mean ± SEM of three independent experiments. (D) Tracks of aca− cells migrating toward buffer alone, 10 nM cAMP, ACA-YFP/aca−, or aca− EVs in the EZ-TAXIScan microfluidic chamber. The images show tracks of individual cells migrating as circles (from red to blue with increasing time) overlaid on the final image. The table indicates the chemotaxis index (CI), speed, and number of tracks of cells that responded to the chemoattractant. Results presented as mean ± SD of three independent experiments. (E) Bar graph showing ATP levels from lysed EV preparations derived from ACA-YFP/aca−, WT, or aca− cells. Results presented as mean ± SEM of three independent experiments. (F) Bar graph showing adenylyl cyclase activity levels in lysed EV preparations derived from aca−/acb− or ACA-YFP/aca− cells 10 min after MnSO4 addition. Results presented as mean ± SEM of three independent experiments. (G) Time-dependent release of cAMP from intact EVs isolated from ACA-YFP/aca− cells in the presence or absence of the adenylyl cyclase inhibitor SQ25536 (60 µM). Results presented as mean ± SEM of three independent experiments. All statistics are shown as mean ± SEM, unless mentioned otherwise. ***, **, *, and ns indicate P < 0.0002, P < 0.002, P < 0.03, and P > 0.12, respectively. Significance testing was performed using unpaired t test corrected for multiple comparison (wherever applicable) using Bonferroni–Dunn method.