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Figure 2 | Molecular Cancer

Figure 2

From: EGFR and EGFRvIII undergo stress- and EGFR kinase inhibitor-induced mitochondrial translocalization: A potential mechanism of EGFR-driven antagonism of apoptosis

Figure 2

EGFRvIII translocates into mitochondria even under unstressed condition but the transport is enhanced by apoptosis inducers and EGFR inhibition. Since endogenous EGFRvIII expression is not maintained in vitro [28], we used U87MG-EGFRvIII stable transfectant cells expressing EGFRvIII [12] in these studies. A, Full-length EGFRvIII is present in the mitochondria under unstressed condition and the level is enhanced by apoptotic stress and Iressa. Cancer cells treated with and without 1 uM staurosporine (ST) and 100 ng/ml anisomycin (AN) for 15 min were fractionated into mitochondrial and non-mitochondrial fractions followed by western blotting. Full-length EGFRvIII was detected in both extracts. The mtEGFRvIII indices indicate that staurosporine and anisomycin increased the levels of mitochondrial EGFRvIII by approximately 3-4 folds. B, Iressa induces significant mitochondrial transport of EGFRvIII. Cancer cells were treated with and without 12.5 uM Iressa, an EGFR kinase inhibitor, for 15 min and subjected to cell fractionation and western blotting. Effectiveness of cell fractionation was indicated by the lack of the cytoplasmic marker β-actin and a nuclear protein lamin B in the mitochondrial extracts, as well as, by the absence of the mitochondrial protein Cox IV in the non-mitochondrial extracts. The mtEGFRvIII indices indicate that Iressa resulted in a marked increase (7.5-fold) of mitochondrial EGFRvIII. C, Immunofluorescence staining and confocal microscopy confirmed staurosporine-, anisomycin and Iressa-induced EGFRvIII mitochondrial import. In these studies, we used an EGFR antibody that recognized the N-terminal epitope in EGFRvIII and the Myc antibody that bound to the C-terminal Myc-tag of the EGFRvIII fusion protein (data not shown) and found both antibodies to detect mitochondrial EGFRvIII. This observation and the molecular weight shown in western blotting suggest that the mitochondrially localized EGFRvIII is highly likely to be the full-length protein.

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