Figure 4.

Nelfinavir induces mitochondria protection despite caspase 8-mediated mcl-1 cleavage. A) The indicated leukemia cell lines were treated with 8 μg/ml nelfinavir for 24 h hours and subjected to PCR analysis to detect potential mcl-1 splice forms and transcriptional mcl-1 regulation. B) An enriched cellular mitochondria fraction of IM9 cells was treated with recombinant caspase 3 or caspase 8 as indicated and subjected to Western blot analysis for mcl-1 expression. Whole cell extracts of nelfinavir-treated IM9 cells were run in parallel for comparison. C) IM9 cells treated for 24 h with 8 μg/ml nelfinavir or 500 nM staurosporine were separated into a crude mitochondria fraction (M) and a crude cytosol fraction (C) and analyzed by Western blot for the distribution of mcl-1, smac and mitochondria-resident protein cytochrome c oxidase (COX IV). For direct comparison, mcl-1 and COX IV immunostaining were performed on the same blot. Staining intensities were analyzed with a BioRad gel documentation system using the BioRad Quantity One program (BioRad, Munich, Germany) and expressed as relative expression values. D) IM9 cells were treated with 10 μg/ml nelfinavir for the indicated times and analyzed by Western blot analysis. Selected bands (mcl1: 43, 36, 32 kDa; caspase 3: 17+19 kDa; caspase 8: 18 kDa) were densitometrically analyzed using the BioRad Quantity One program and plotted as relative expression values versus β-actin expression.