The cytotoxicity effect of Z-LLNle-CHO could be reversed by edaravone that blocks its proteasome inhibitory function. (a) Cells were treated with indicated drugs for 72 hours before cell growth was measured using MTS assay. Results represent the mean ± standard deviation (SD) of three independent experiments. (b) Proteasome activity in intact cells was directly measured using a cell-based assay after cells were treated with different combinations of drugs for two hours. The treatment conditions were (from left to right): dimethyl sulfoxide (DMSO) vehicle only; bortezomib alone; bortezomib plus tiron; bortezomib plus edaravone; Z-LLNle-CHO alone; Z-LLNle-CHO plus tiron; and Z-LLNle-CHO plus edaravone. The concentrations of bortezomib, tiron, edavarone, and Z-LLNle-CHO are 100 nM, 2 mM, 100 μM, and 5 μM for MCF-7 cells, and 40 nM, 0.5 mM, 100 μM, and 2.5 μM for MDA-MB-231 cells, respectively. Results represent the mean ± SD of three independent experiments. (c) The same protein samples used for immunoblotting in Figure 3b plus another negative control sample were subjected to immunoblotting with anti-Notch1 (V1744) antibody that specifically recognizes active Notch1 intracellular domain. The order of the samples were the same as that in Figure 3b except that lane 1 is the new negative control sample.