Cells were challenged for 90 min with purified Ply (+Ply) at 250 nM or at the indicated concentration. a, wild-type asytrocytes. b-f, HeLa cells or stable transfectants expressing Cx43 (HCx43). Incubation in the presence of: +HEX: hexokinase; + CBX: 100 μM Carbenoxolone. a, b, Cells were fixed and processed for the quantification of nuclear (a) or cell retraction (b). Median values are represented. > 25 cells per sample. Mann-Whitney. ****: p < 0.001. d, > 72 cells per sample. ANCOVA. ****: p < 0.001. c, d, calcein-loaded cells were incubated with 250 nM Ply. c, representative experiment of Ply-induced calcein release. d, rates of calcein decrease determined from linear fits as in “c” (Materials and Methods). Bars: median values. Mann-Whitney. ****: p < 0.001.