Figure 6

Oncogenic signal transduction downregulation (I). Geldanamycin-induced Hsp90 inhibition results in functional impairment of Akt-dependent signaling in human urinary bladder cancer cells. (A) Western blotting experiments reveal drastic downregulation of Akt signaling activity, evolving through the IGF-IR/Akt/IKK signal transduction axis, as evidenced by the eradication of both total and constitutively phosphorylated protein forms of all examined Hsp90 protein clients (IGF-IR, Akt, IKKα and IKKβ), in RT4 and T24 bladder cancer cell lines. Actin was used as protein of reference. All Western blottings were carried out three times, with a representative image collection shown here. (B) Protein densitometric quantification bars, denoting the drug-induced expression level alterations of the IGF-IR/Akt/IKK signaling axis components, and their respective phosphorylated forms, compared to control conditions, using Actin (A) as protein of reference. Standard deviation values are depicted as error bars on top of each value. (C) Immunofluorescence images illustrating the cellular localization of Hsp90 (red color) and Akt kinase (green color) in RT4 and T24 bladder cancer cells, in the presence or absence of geldanamycin. Upon drug administration (10 μΜ), a pronounced depletion of cytoplasmic Akt protein is detected, in contrast to what is observed under control conditions. The bright orange color (white arrows) indicates the cellular areas where Akt and Hsp90 are likely co-localized (associated), in the absence of drug conditions. Images were taken under a Nikon EZ-C1 confocal microscope. All immunofluorescence experiments were conducted three times, while a characteristic image collection is presented here (scale bars: 10 μm).