EGFR and EGFRvIII puma fierce both exist in complex with PUMA constitutively and the interactions are sustained under apoptotic stress. U87MG-EGFR and U87MG-EGFRvIII stable transfectants and T98G GBM cells with endogenous EGFR were treated with 1% DMSO and 1 uM apoptosis-inducer staurosporin (ST), harvested and subjected to immunoprecipitation/western blotting (left panel) and western blotting (right panel), as described earlier.

Lack of ±-tubulin and Cox IV in the mitochondrial and non-mitochondrial fractions, respectively, indicated fractionation effectiveness. Low mtPUMA indices indicate that PUMA is primarily localized in the cytoplasm of U87MG-EGFRvIII cells, under unstressed and stressed conditions. (B) PUMA is exclusively rihanna puma creepers localized in the mitochondrial fractions of U87MG-vector cells. U87MG-vector cells were treated, fractionated and proteins analyzed, as described earlier in panel a .

The mtPUMA indices in these cells are 1.0, indicating fenty puma slides that PUMA is exclusively detected on the mitochondria of these cells, independent of apoptotic stress. (C) EGFR expression knockdown by siRNA leads to increased PUMA mitochondrial translocalization. In control siRNA-treated T98G natural GBM cells, PUMA is primarily localized in the cytoplasm. In EGFR-specific siRNA-treated cells, we observed a marked increase of mitochondrial PUMA (left panel).

Importantly, siRNA-mediated EGFR expression knockdown led to a puma ignite significant increase (16-fold) of mitochondrial PUMA ( Fig. 5c-left ), further suggesting the ability of EGFR to modulate PUMA mitochondrial translocalization. EGFR-specific siRNA was effective in reducing EGFR expression as shown by the western blots ( Fig. 5c-right ). In addition to GBM cells, we found PUMA to be localized in the cytoplasm of MDA-MB-468 human breast cancer cells .

That are known to express significant levels of endogenous EGFR [ 34 ]. Collectively, these results demonstrate that PUMA is sequestered in the cytoplasm of EGFR- and EGFRvIII-expressing GBM and breast cancer black puma cells, constitutively and under apoptotic stress.To gain insight into the factors that modulate the interaction of EGFR with PUMA, we examined the requirement of EGFR activation for the EGFR-PUMA interaction.

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.