NPI-0052 enhances apoptosis induced by TNF and chemotherapeutic drugs. (A) KBM-5 cells (10 000 cells/0.1 mL) were incubated at 37°C with 1 nM TNF, 10 μg/mL thalidomide, or 20 nM bortezomib in the presence and absence of 50 nM NPI-0052 as indicated for 18 hour, and the viable cells were assayed using the MTT reagent. The results are expressed as mean cytotoxicity (± standard deviation [SD]) from triplicate cultures. (B) Cells (10⁶/mL) were pretreated with 50 nM NPI-0052 for 4 hours and then incubated with 1 nM TNF, 10 μg/mL thalidomide, or 20 nM bortezomib for 16 hours. Cell death was determined by the calcein-AM based live/dead assay as described in “Live and dead assay.” Data are representative of 3 independent experiments showing similar results. (C) U266 (10⁶/mL) or DU145 (10⁵/mL) cells were pretreated with 50 nM NPI-0052 for 4 hours and then incubated with 10 μg/mL thalidomide or 20 nM bortezomib for 16 hours. Cell death was determined by the calcein-AM based live/dead assay as described in “Live and dead assay.” Data are representative of 3 independent experiments showing similar results. Images were acquired as described in “Live and dead assay.” (D) Cells were pretreated with 50 nM NPI-0052 for 4 hours and then incubated with 1 nM TNF for 18 hours. Afterward, they were incubated with anti-annexin V antibody conjugated with FITC plus PI and analyzed with a flow cytometer for apoptotic effects. (E) Cells were pretreated with 50 nM NPI-0052 for 4 hours and then incubated with 1 nM TNF for 18 hours. Cells were fixed, stained with TUNEL assay reagent, and then analyzed with flow cytometer. (F) Cells were pretreated with 50 nM NPI-0052 for 4 hours and then incubated with 1 nM TNF for the indicated times. Whole-cell extracts were prepared and subjected to Western blot analysis using anti-PARP antibody.