Fig. 1: Colon carcinoma cells release vesicles upon P2X7 activation.

A CT26 CRC cells were stained with PKH26GL (red) and quinacrine (green) fluorescent dyes. Images were acquired using confocal microscopy at time 0 and after 5 min following P2X7 activation with 300 µM BzATP and are extrapolated from a 30-min time course (see supplementary videos 1 and 3). B CT26 cells were pre-treated with P2X7 antagonist AZ10606120 (5 µM) for 10 min before application of BzATP. C Number of vesicles released in 30 min from CT26 cells in PBS vehicle (PBS S-VS), following stimulation with P2X7 agonist ATP (P2X7-VS) or 10 min pretreatment with P2X7 antagonist AZ10606120 followed by stimulation with 3 mM ATP (AZ-VS) (n = 5). D Size of PBS S-VS, P2X7-VS, and AZ-VS (n = 5). E Number of vesicles released in 30 min from CT26 cells in DMSO vehicle (DMSO S-VS), following stimulation with P2X7 agonist ATP (P2X7-VS) or 10 min pretreatment with P2X7 antagonist A740003 followed by stimulation with 3 mM ATP (A74-VS) (n = 7). F Size of DMSO- S-VS, P2X7-VS, and A74-VS (n = 7). G Western blot for GM130, Alix, P2X7, CD39, CD73, and A2A in CT26 cells, S-VS and P2X7-VS. Pericellular ATP was measured with the pmeLUC probe expressed on the cell surface of untreated CT26 cells or after 5 min of exposure to PBS vehicle, S-VS, and P2X7-VS (n = 4). H Quantification of luminescence changes was expressed as a fold increase on time 0. I Representative images of photon emissions. Changes in ATP J concentration increase on time 0 in the supernatants of CT26 cells, untreated or treated with PBS vehicle, S-VS, or P2X7-VS, measured with a luciferin/luciferase assay (n = 3). Changes in adenosine K concentration increase on time 0 in the supernatants of CT26 cells untreated or treated with PBS vehicle, S-VS, P2X7-VS, or P2X7-VS plus 5uM CD73 inhibitor AB680 (n = 5). *p < 0.05, **p < 0.001, ***p < 0,0001, ****p < 0.00001.