(A) Siomycin A stabilizes protein expression similarly to proteasome inhibitor MG132. U2OS cells treated as indicated were harvested and immunoblotting was carried out with antibodies specific for p53, p21, hdm2, Mcl-1, and β-actin. (B) Thiazole antibiotics inhibit NF-kB transcriptional activity. 293T-NF-κB-Luc cells were induced with 10 ng/mL TNF-α for 24 hrs and treated with 5 µM of the thiazole antibiotics for additional 10 hrs followed by luciferase assay. The results shown are mean±SD of three separate experiments. (C) Inhibition of 20S proteasome activity by Siomycin A and thiostrepton. The inhibition of proteasome activity by the thiazole antibiotics in vitro was less efficient compared with proteasome inhibitors, MG-132 and lactacystin as detected by using 20S Proteasome activity kit (Millipore). The free AMC fluorescence was quantified at 380/460 nM in a fluorometer (SpectraMax GeminiXS, Molecular Devices). The results shown are mean±SD of three separate experiments. (D) Proteasome inhibitors inhibit FoxM1 transcriptional activity. C3-Luc cells were treated with a combination of 1 ug/ml doxycycline and the indicated concentrations of the proteasome inhibitors MG115, MG132 and bortezomib (BOR). The luciferase activity was determined by using the Luciferase Assay System (Promega) according to the manufacturer's instructions. The results shown are mean±SD of three independent experiments. The asterisks on each graph represent statistically significant differences with p values<0.001.

(A) Proteasome inhibitors induce apoptosis and inhibit FoxM1 protein expression in multiple myeloma, leukemia and osteosarcoma cell lines. U266 and RPMI8226 multiple myeloma, HL-60 leukemia and U2OS-C3 osteosarcoma cell lines were treated with proteasome inhibitors MG115, MG132 and bortezomib (BOR). Apoptosis was assessed by immunoblotting for cleaved caspase-3 and FoxM1 protein levels were detected by immunoblotting. β-actin was used as the loading control. (B) Proteasome inhibitor-induced apoptosis was further quantified by using Annexin V -PE/7AAD staining. U266 and RPMI8226 multiple myeloma, HL-60 leukemia and U2OS-C3 osteosarcoma cell lines were grown in the absence or presence of indicated concentrations of proteasome inhibitors MG115, MG132 and bortezomib (BOR) for 24 hours. Following drug treatment cells were stained with Annexin V- PE/7AAD and then analyzed by flow cytometry. Percentage of apoptotic cells is shown in brackets. Concentrations of drugs are the same as in the Fig 2A. (C) Overexpression of FoxM1 specifically protects against cell death induced by Bortezomib. Overexpression of FoxM1 protected against cell death induced by increasing amount of bortezomib (BOR), but not that of doxorubicin. Immunoblotting was carried out with antibodies specific for cleaved caspase-3 and β-actin.