EGFR undergoes enhanced mitochondrial translocalization after exposures to apoptosis inducers and the EGFR kinase inhibitor, Iressa. A, An apoptotic inducer and an EGFR kinase inhibitor enhance EGFR mitochondrial transport. Human T98G GBM cells with high endogenous levels of EGFR were treated with and without 1 uM staurosporine (ST) and 12.5 uM Iressa (I) for 15 min, fractionated into mitochondrial and non-mitochondrial fractions, and analyzed by western blotting. The extent of EGFR mitochondrial translocalization was indicated by mtEGFR index. Right panel: western blots with whole cell lysates. B, Immunofluorescence staining/confocal microscopy showing staurosporine- and Iressa-induced EGFR mitochondrial translocalization. T98G cells were similarly treated as in Panel A, fixed and subjected to immunofluorescence staining/confocal microscopy to mark mitochondrial EGFR. Mitochondrial EGFR (arrows) is shown as the yellow signals merged from the green fluorescence (EGFR) and the red fluorescence (mitotracker, a mitochondria-specific dye). Bottom panels: high-resolution images. C, Endogenous EGFR in breast cancer MDA-MB-468 cells undergoes staurosporine-induced mitochondrial transport. The cells were treated with and without staurosporine (ST) for 15 min, fractionated and subjected to western blotting. Right panel shows western blots with whole cell lysates. D, Anisomycin stimulates EGFR mitochondrial translocalization in MDA-MB-468 cells. The cells were treated with and without 100 ng/ml anisomycin for 15 min and subjected to immunofluorescence staining/confocal microscopy, as described earlier in Panel B. Arrows: representative EGFR signals in the mitochondria. E, EM shows mitochondrial presence of EGFR in EGF-treated tumor cells. Arrows point to EGFR signals in the mitochondria. Right panels show high-resolution images. Mitochondrion is labeled as M. Nucleus is labeled as Nu. F, Staurosporine and Iressa induced EGFR mitochondrial transport in a time-dependent fashion peaking at 2 hrs after treatments. The majority of T98G cells did not survive 24 hr staurosporine treatment as indicated by the lack of β-actin and EGFR expression.