Figure 5

Figure 5

Depletion of reduced glutathione (GSH) and induction of high-molecular-weight complexes in AML cells. (A) GSH levels in AML cells after different times of exposure to 0.35 μM VLX1570 and/or 10 μM BSO. Means and SDs from one representative experiment are shown; (B) fraction viable cells determined by the AO/PI method after treatment with VLX1570 and/or BSO. Means and SDs from one representative experiment are shown; (C) (top) high molecular weight (HMW) region of Coomassie BB-stained SDS-PAGE gels loaded with extracts from cells exposed for 4 h to different concentrations of VLX1570, (bottom) immunoblots of extracts from VXL1570-exposed cells probed with an antibody to K48-linked polyubiquitin, note the distribution of high molecular weight bands marked with yellow and orange dots; (D) effect of 10 μM BSO on the formation of HMW complexes and loss of HMW proteins in KG1a cells exposed to 0.5, 1, 2, and 5 μM VLX1570; (E) KG1a cell extracts were exposed to 1 μM VLX1570 for increasing times at 37 °C and analyzed by SDS-PAGE; (left): gel stained with Coomassie blue, (right): immunoblot probed with an antibody to K48-linked polyubiquitin; and (F) effect of pretreatment of cell extracts with 20 μM NEM, 100 μM Tiron, or the Michael-acceptor-containing compound CB360 (30 μM) [40] on the formation of HMW complexes. Plots (A,B) show the average of three replicates, and error bars represent one standard deviation.