Z-LLNle-CHO has a proteasome inhibitory function. (a) Proteasome activity in intact cells was directly measured using a cell-based assay after MCF-7 and MDA-MB-231 cells were treated with indicated drugs for two hours. Results represent the mean ± standard deviation of three independent experiments. (b) Protein samples were prepared from MCF-7 and MDA-MB-231 cells that were treated with different combinations of drugs overnight and were subject to immunoblotting with anti-ubiquitin antibody (clone FK2 from Millipore, 1:1,000). Actin immunoblotting was used as loading control. The treatment conditions were (from lane 1 to lane 8): dimethyl sulfoxide (DMSO) vehicle only; Z-LLNle-CHO alone; Z-LLNle-CHO plus tiron; Z-LLNle-CHO plus edaravone; bortezomib alone; bortezomib plus tiron; bortezomib plus edaravone; lactacystin. The concentrations of Z-LLNle-CHO, tiron, edavarone, bortezomib, and lactacystin are 5 μM, 2 mM, 100 μM, 100 nM, 20 μM for MCF-7 cells, and 2.5 μM, 0.5 mM, 100 μM, 40 nM, 5 μM for MDA-MB-231 cells, respectively. The accumulation of polyubiquitinated proteins is an indicator of proteasome inhibition. (c) MCF-7 and MDA-MB-231 cells were treated with different combinations of drugs for four hours and then immunostained with anti-ubiquitin FK2 antibody. The treatment conditions were (from 1 to 8): DMSO vehicle only; Z-LLNle-CHO alone; Z-LLNle-CHO plus tiron; Z-LLNle-CHO plus edaravone; bortezomib alone; bortezomib plus tiron; bortezomib plus edaravone; DAPT. The concentrations of Z-LLNle-CHO, tiron, edaravone, and bortezomib were the same as that were used for preparation of protein samples in subsection b. 5 μM of DAPT was used for both MCF-7 and MDA-MB-231 cells. The redistribution of nuclear ubiquitin to cytoplasm is a phenomenon that can be induced by proteasome inhibition.