FIG. 9.
MG115 sensitizes primary keratinocytes upstream or at the mitochondrial level. (A) Cytochrome c, Smac/DIABLO, and HtrA2/Omi release after TRAIL treatment of primary keratinocytes. Cells were treated for the indicated time intervals with 50 ng of TRAIL per ml in the presence or absence of 10 μM MG115. Cytoplasmic lysates were subsequently prepared as described in Materials and Methods and analyzed for cytochrome c, Smac/DIABLO, and HtrA2/Omi by Western blot analysis. Equal loading of cytoplasmic proteins was determined by reprobing of blots with antitubulin antibodies. (B) TRAIL-induced loss of mitochondrial transmembrane potential ΔΨm requires the presence of the proteasome inhibitor MG115. Cells were incubated for 60 min with either diluent alone or 10 μM MG115, followed by treatment with 50 ng of TRAIL per ml. Cells were analyzed for ΔΨm by staining with 40 nM tetramethylrhodamine ethyl ester 4 h later. Quantitative disruption of ΔΨm was only seen in cells treated with TRAIL in the presence of the proteasome inhibitor MG115.