P2X7 Receptor-Dependent Blebbing and the Activation of Rho-Effector Kinases, Caspases, and IL-1 Release

Files in this Data Supplement:

• Movie 1. (AVI format, 2435KB) - BAC1 Macrophages Demonstrate ATP-induced Tethered Blebbing. Macrophages were treated as described for Figure 2. Briefly, macrophages were equilibrated in BSS and treated with 3 mM ATP at the 5:00 time-point in the recordings at 37ºC, in the presence of 1 µM YoPro. Note the dynamic extrusion and retraction of tethered blebs.
• Movie 2. (AVI format, 1786KB) - HEK-P2X7 Cells Demonstrate ATP-induced Zeiotic Blebbing. HEK293 cells stably expressing the human P2X7 receptor were treated as described for Figure 3. The HEK-P2X7 cells were equilibrated in BSS and treated with 3 mM ATP at the 5:00 time-point in the recordings, at 37ºC and in the presence of 1 µM YoPro. Note the dynamic zeiotic blebbing which is morphologically distinct from the tethered blebbing observed in BAC1 macrophages. Also, HEK-P2X7 cells initiate blebbing more rapidly than BAC1 macrophages.
• Movie 3. (AVI format, 2139KB) - Effect of a ROCK Inhibitor on Blebbing in HEK-P2X7 Cells. HEK-P2X7 cells were treated as described for Figure 4. The cells were pre-incubated with 10 µM Y-27632 to block ROCK activity; 3 mM ATP was subsequently added at the 5:00 time-point in the video recording, in the presence of 1 µM YoPro at 37ºC. Note the complete absence of blebbing, yet YoPro dye fluorescence is still clearly visible.
• Movie 4. (AVI format, 2569KB) - Effect of a ROCK inhibitor on Blebbing in BAC1 Macrophages. BAC1 macrophages were treated as described for Figure 5. Macrophages were pre-incubated with 10 µM Y-27632 to block ROCK activity, followed by 3 mM ATP treatment at the 5:00 time-point in the video. No blebbing can be observed, and furthermore, the cells have an unusual morphology in which it appears they are unable to retract exuded cytoplasm; compare with S2.