FIGURE 3.

Chemotherapeutic drugs induce accumulation of active pannexin-1 channels via a caspase-dependent activation mechanism. A, schematic of the YoPro dye uptake end point assay. Jurkat T cells were incubated as follows: with no stimulus for 4 h (B); with 250 ng/ml anti-Fas, 4 h (C); 3 μm STS, 4 h (D); 20 μm Etop, 8 h (E); or 25 μm Dox (F) in the absence or presence of 100 μm Z-VAD. The treated cells were then washed, resuspended in basal saline supplemented with 1 μm YoPro ± 100 μm CBX, and incubated for 20 min prior to plate reader quantification of accumulated YoPro fluorescence per well (G) or phase-contrast and epifluorescence imaging (B–F). G, data indicate mean ± S.E. for n = 3 experiments for STS, Etop, and Dox, and n = 2 experiments for anti-Fas. *, p < 0.05; **, p < 0.01; ***, p < 0.001. H, schematic of the YoPro dye uptake kinetic assay. Jurkat cells were suspended in basal saline supplemented 1 μm YoPro ± 100 μm CBX ± 100 μm Z-VAD transferred to the wells of a 24-well plate. Fluorescence (485/540 nm) was measured at 1-min intervals for 15 min prior to addition of 3 μm STS (or vehicle) and then at 1-min intervals for an additional 4 h prior to the addition of digitonin (Dig) to permeabilize the cells. Data are representative of three experiments.