Fig. 5.

Nuclear-localized PDK1 protects human prostate tissue-derived cell lines from apoptosis. (A) Representative confocal images showing subcellular localization of PDK1 in LnCAP, PC-3, and DU145 cancer cells. LnCAP, PC-3, and DU145 cells growing on coverslips were fixed and stained with an anti-PDK1 antibody (green). The nuclei were visualized by staining with DAPI (blue). n = 4 experiments. (B) Comparison of PI3K pathway activity between DU145 and LnCAP cells lines. Note the slower mobility of endogenous PDK1 (marked with an asterisk) in LnCAP cells in which most PDK1 were found in the nucleus [see (A)]. (C) Responses of DU145 and LnCAP cells to apoptotic stimuli. Cells were treated for 6 hours with TNF-α (5 ng/ml) and actinomycin D (100 ng/ml), UV-C (30 mJ/m²), or doxorubicin (1 μg/ml) and lysed. Apoptosis was determined by Western blotting analysis with antibodies against the indicated proteins. (D) Retrovirus-mediated expression of nuclear PDK1 protects DU145 cells from TNF-α- stimulated JNK1 activation and apoptosis. Retroviral expression of empty vector or vector expressing nuclear PDK1 and subsequent treatment with TNF-α were performed as described in Materials and Methods. TNF-α and JNK activation and apoptosis were measured in cell lysates as in (C).