Effect of PEITC on MCL1 stability and its glutathionylation state in CLL cells. (A) Time-dependent effect of 5 μM PEITC on MCL1 and BCL2 protein levels in F-ara-A–sensitive and –resistant CLL cells, detected by Western blot analysis. (B) Quantitation of MCL1 protein after exposure to PEITC as described in panel A. Each bar represents the mean and 95% CI from 7 F-ara-A–sensitive or 6 of F-ara-A–resistant CLL patient samples. (C) Suppression of PEITC-induced MCL1 degradation by caspase inhibitor Z-VAD-fmk. Cells were pretreated with 20 μM Z-VAD-fmk for 30 minutes before exposure to 5 μM PEITC. A representative Western blot from experiments with a CLL sample is shown. Similar results were obtained using another sample. (D) Suppression of PEITC-induced MCL1 degradation by NAC. CLL cells were preincubated with 1 mM NAC for 1 hour before exposure to 5 μM PEITC for 5 hours. Each bar represents the mean and 95% CI of 4 different CLL samples. (E) Glutathionylation prevented caspase-3–mediated cleavage of MCL1 in vitro. Dialyzed CLL lysates were incubated with 0.5 mM NADH, 0.5 mM NADPH, 2 mM GSH, or 2 mM GSSG for 10 minutes, and then exposed to recombinant caspase-3 for 60 minutes. Cleavage of MCL or PARP was detected by Western blot analysis. Representative results are shown. (F) PEITC treatment reduced the level of glutathionylation of MCL1 in CLL cells. CLL cells were exposed to 5 μM PEITC for 2.5 hours. Glutathionylated protein was immunoprecipitated (IP) using anti-GSH, and the levels of glutathionylated MCL-1 were analyzed using immunoblotting (IB) with anti-MCL1 antibody. Because PEITC induces rapid degradation of MCL1 protein, 3-fold higher amount of protein from the PEITC-treated sample was used for IP (input) to allow the detection of MCL1 signal. (G) Depletion of total glutathione and ratio of GSH/GSSG after 5 μM PEITC treatment as in panel F. Addition of 1 mM NAC restored cellular glutathione. Each stacked bar (GSH/GSSG) represents the average of duplicate measurements.