Figure 2.

IRX-2 blocks MV-induced activation of caspases-3 and -7 and a loss of mitochondrial membrane potential (MMP) in CD8⁺ Jurkat cells. (a) Activation of caspases-3 and -7 in CD8⁺ Jurkat cells was assessed by flow cytometry for caspase-3/-7-FAM binding. Results of a representative experiment out of five experiments performed are shown. (b) Mean fluorescence intensity (MFI) of caspase-3/-7-FAM expression was determined in CD8⁺ Jurkat cells that were either untreated, incubated with 10 μg MV for 3 h or pretreated for 24 h with IRX-2 (1 : 3 dilution) and then incubated with MV for 3 h. CD8⁺ Jurkat cells were also co-incubated with MV and 20–100 μM of the pan-caspase inhibitor Z-VAD-FMK (zVAD) or co-incubated with MV and the anti-Fas neutralizing mAb ZB4 (10 μg/ml) (controls). Results are the mean MFI± S.D. of three independent experiments. (c) Western immunoblots showing caspase-3 activation in CD8⁺ Jurkat cells. The cells were either untreated, treated with IRX-2 (1 : 3 dilution) for 24 h (+ IRX), treated with MV (10 μg) for 3 h (+ MV 3 h) or 24 h (+ MV 24 h) or preincubated with IRX-2 for 24 h and then treated with MV (10 μg) for 3 or 24 h (+ IRX→MV). Whole-cell lysates of the cells were separated on SDS-PAGE and transferred to PVDF membranes for subsequent western blotting. Activation of caspase-3 is shown as a decrease in the inactive proform and the appearance of the active subunits p17 and p10. Results are representative of three independent western blots. (d) CD8⁺ Jurkat cells were analyzed by flow cytometry for a decrease in red fluorescence of the cationic dye, JC-1, indicating a loss of MMP. Percentages of JC-1-red-negative cells were determined in untreated CD8⁺ Jurkat cells, after 3 h treatment with MV (10 μg) alone or after 24 h pretreatment with IRX-2 (1 : 3 dilution) +MV. Cells treated with 50 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a protonophore that dissipates the H⁺ gradient across the inner mitochondrial membrane, were used as a positive control. The data are from a representative flow cytometry experiment out of five experiments performed. (e) Effects of MV alone, IRX + MV or MV in combination with Z-VAD-FMK or ZB4 on a loss of MMP in Jurkat cells. The data are means±S.D. of five independent experiments (*P<0.005; **P<0.002 compared to samples without IRX-2)