Figure 5

DMC more potently inhibits proteasomal activity than curcumin. (a) MDA-MB 435S cells treated with the indicated concentrations of DMC or curcumin for 24 h were subjected to western blotting. Protein poly-ubiquitination and free ubiquitin levels were examined by western blot analysis using an anti-ubiquitin antibody. β-Actin was examined to verify equal loading (left). Quantitation of the free ubiquitin and ubiquitinated protein levels. Compared with control (untreated cells), the fold change of protein levels was determined. Two independent western blots were normalized to β-actin band in the same sample (right). (b) MDA-MB 435S cells treated with 20 μ M DMC or curcumin for 16 h were fixed, immunostained using anti-ubiquitin antibody (green) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Processed cells were observed under the fluorescence and phase contrast microscope. Bar, 20 μm. (c) Cellular proteasomal activity in MDA-MB 435S cells treated with curcumin or DMC. MDA-MB 435S cells were treated with curcumin or DMC at the indicated concentrations for 6, 12 and 24 h. The activities of 20S proteasome, including trypsin-like, chymotrypsin-like and PGPH-like activities, in the cell lysates were measured as described in Materials and Methods section. (d) The effects of curcumin or DMC on proteasome hydrolytic activities. Purified 20S proteasome (70 ng) was incubated with the indicated concentrations of curcumin or DMC for 2 h. Proteasomal trypsin-like, chymotrypsin-like and PGPH-like activities were measured as described in Materials and Methods section. (e) MDA-MB 435S cells treated with 20 μ M DMC for the indicated time points were subjected to western blotting of the indicated proteins. Western blotting of β-actin was served as a loading control. (f) MDA-MB 435S cells treated with the indicated concentrations of DMC or curcumin for 24 h and cell extracts were prepared for western blotting of the indicated proteins