ÿþSimilar to the PUMA down-regulation, over-expression of puma creepers EGFRvIII in U87MG cells resulted in significant resistance to anisomycin-induced apoptosis ( Fig. 3c,d ). After treatment with anisomycin, there was only 3.1% apoptosis in U87MG-EGFRvIII cells. In contrast, the isogenic low EGFR expressing U87MG-vector cells underwent massive apoptosis (49.8%), similar to the level observed with PUMA down-regulation (45.5%). The results in Fig.

The mitochondrial fractionation/western blotting ( Fig. 5a ) showed PUMA to be primarily localized in the non-mitochondrial fractions of U87MG-EGFRvIII cells under unstressed condition and to undergo a modest mitochondrial translocalization following exposure to the apoptotic inducers, staurosporin (ST) puma fenty and anisomycin (AN). As indicated by the low mtPUMA index (0-0.02), the majority of PUMA is sequestered in the non-mitochondrial fractions of the EGFRvIII-expressing U87MGEGFRvIII cells ( Fig. 5a ).

The extent of puma slides PUMA mitochondrial translocalization, mtPUMA Index, is computed as described earlier in Materials and Methods. (A) PUMA is primarily localized in the cytoplasm of U87MG-EGFRvIII cells. U87MG-EGFRvIII cells treated with vehicle control, staurosporin (ST, 1 uM) or anisomycin (AN, 100 ng/ml) were harvested and fractionated into mitochondrial and non-mitochondrial fractions. Protein extracts from both fractions were subjected to western blotting to detect PUMA.

PUMA is primarily localized in puma rihanna the cytoplasm of these cells, under unstressed and stressed conditions, as indicated by the low mtPUMA indices.Similar observations were further found in T98G GBM cells that naturally express EGFR ( Fig. 5c ). The modest detection of mitochondrial PUMA in these cells may potentially be the result of insufficient cytoplasmic EGFR to interact with and sequester all the PUMA molecules in the cytoplasm.

Fig. 6a (left panel) shows that the ability of EGFR to bind to PUMA was similar in U87MG-EGFR cells with and without EGF stimultion following serum starvation. As indicated by the absence of auto-phosphorylated EGFR puma suede classic (p-EGFR, Y1068), serum-starved U87MG-EGFR cells express inactive EGFR ( Fig. 6a-right ). In contrast, p-EGFR is readily detected in EGF-treated cells, indicating that EGF efficiently activated EGFR in these cells.

U87MG-EGFR cells were serum-starved for 24 hrs and stimulated with EGF for 20 mins. The cells were harvested and subjected to immunoprecipitation/western blotting (left panel) and western blotting (right panel). An EGFR antibody was used to immunoprecipitate EGFR. Control IgG did not yield signals indicating specificity. Auto-phosphorylated Y1068 residue serves as an indicator for EGF-induced EGFR activation. (B) EGFR kinase activity is not required for their interaction with PUMA.