MIPP-induced vacuolation leads to non-apoptotic cell death in glioblastoma cells. A) MTT assay of U251 cells treated over time with the indicated compounds (refer to Fig. 1 for structures) at a concentration of 10 μM. Each point represents the mean (± SD) of results from quadruplicate wells. The decreases in viability of the cells treated with MIPP (▲) or compound I (●) were significant at p < 0.001 on days 2 and 3. B) Phase-contrast images of U251 cells treated for 2 or 3 days with 10 μM MIPP. The arrows point to vacuolated cells that have rounded and detached from the surface of the dish. The scale bar is 10 microns. C) U251 cells were treated with 10 μM MIPP or an equivalent volume of DMSO (control) for 2 days and colony forming assays were performed as described in Materials and Methods. The results shown are the mean (± SD) of triplicate dishes, with representative dishes shown at the right. The decrease in the number of colonies for the MIPP-treated cells (*) was significant at p < 0.001. D) U251 cells were examined by electron microscopy after two days of treatment with 10 μM MIPP. The left panel (att) is a representative image of an attached cell and the right panel (det) is representative of a cell that had detached from the dish. The arrows point to regions of plasma membrane discontinuity indicative of cell rupture. Nuclei (N) do not show changes in chromatin distribution typical of apoptosis. The scale bars are 10 microns. E) U251 cells treated with 10 μM MIPP for 2 days are negative for TUNEL staining. F) Inhibition of caspase activity does not prevent MIPP-induced cell death. U251 cells were seeded at 350,000 cells per 60 mm dish. The next day cells were treated with 10 μM MIPP or an equivalent volume of DMSO, in the presence or absence of 50 μM z-VAD-fmk. After two days the attached and detached cells were pooled and harvested for immunoblot analysis of PARP. MTT assays were performed on cells treated in the same manner, except that they were seeded in a 96-well plate. Values are means (± SD) of quadruplicate samples.