Figure 8.
NSCLC cells selected for resistance to obatoclax display a block in apoptosis. (a) H460 NSCLC cells were selected for resistance to obatoclax (H460-ENU) using ENU mutagenesis as described in the Materials and Methods. (left) H460-ENU cells displayed a significant increase in their EC50 value as determined by ViaLight assay. (right) H460 parental (H460par) cells and H460-ENU cells were seeded as previously described for clonogenic assay and treated with indicated doses of obatoclax to assess the effect of obatoclax on clonogenic growth. (b) H460-ENU cells are resistant to obatoclax-induced apoptosis and display reduced LC3 processing. H460par and H460-ENU cells were treated with indicated doses of obatoclax for 48 h and the level of PARP, caspase 9 cleavage and LC3 processing was determined by SDS–PAGE. β-Actin was used as a loading control. (c) H460-ENU cells displayed a block in SMAC and cytochrome c release. H460 and H460-ENU cells were treated with 500 nM obatoclax for 48 h. The cytosolic fraction of cells was isolated as described in Materials and methods and the levels of SMAC and cytochrome c determined by SDS–PAGE. β-Actin was used as a loading control