Fig. 3.

ITC-induced proteasome inhibition is unrelated to protein aggregation and ROS generation. Pretreatments with colchicine (A), vinblastine (B) and paclitaxel (C) did not have substantial effects on proteasome inhibition by BITC. HeLa cells were pretreated with 1 or 10 μM colchicine, vinblastine or paclitaxel for 1 h followed by treatment with 10 μM BITC for another 4 h. White bars: chymotrypsin-like activity; striped bars: trypsin-like activity; black bars: caspase-like activity. (D) Proteasome was inhibited by 1 mM H2O2 or 10 μM PEITC. U266 cells were treated with various concentrations of H2O2 or 10 μM PEITC for 4 h. (E) Polyethylene glycol-linked catalase did not affect ITC-induced proteasome inhibition. U266 cells were pretreated with 500 U (low cat., lower amount of catalase) or 1000 U (high cat., higher amount of catalase) polyethylene glycol catalase for 1 h followed by 10 μM BITC for 4 h. (F) 3-Amino-1,2,4-triazole (ATZ), a catalase inhibitor, did not aggravate BITC-induced proteasome inhibition. U266 cells were treated with 10 μM ATZ alone or in combination with 10 μM BITC for 4 h. White bars: chymotrypsin-like activity; striped bars: trypsin-like activity; black bars: caspase-like activity. (G) Three ITCs depleted GSH at similar rates. U266 cells were treated with 10 μM (white bars) and 20 μM (black bars) of BITC, PEITC, SFN and NMPEA for 1 h. Cellular GSH concentration was measured and the relative amount was calculated against the dimethyl sulfoxide-treated control. *P < 0.01; **P < 0.001.