Figure 3. Molecular signature of NVP-AUY922 in combination with radiation.

(A) To confirm depletion of HSP90 client proteins linked to radioresistance, levels were assessed in vitro in HeLa and HN3 cell lines by western blot. Cells were treated with vehicle only, or the concentrations of NVP-AUY922 indicated for 24 h, followed by reducing SDS-PAGE of 30 µg whole cell lysate and western blot detection of the HSP90 client proteins shown. (B) Levels of HSP72 28 h post NVP-AUY922 treatment and phospho-ErbB2 24 h post NVP-AUY922 treatment were determined by densitometry of three independent experiments (see Fig. S2A). (C) HeLa and HN3 cells were pre-treated with NVP-AUY922 for 24 h at the concentrations indicated. Cells were irradiated with 4 Gy or 10 Gy and 4 h later whole cell lysates harvested and probed for HSP72 by western blot. (D) Cells were pre-treated with 10 nM NVP-AUY922 for 24 h with 4 Gy of radiation delivered at the 0 hour time point. At the subsequent time points indicated, whole cell lysates were harvested and probed for total-ErbB2, phospho-histoneH3 and HSP72. (E) Tumor samples were harvested at the point of final irradiation to determine response to NVP-AUY922 in vivo. Tissue samples were homogenised and 30 µg of protein probed for HSP72 by reducing SDS-PAGE and western blot. γ-tubulin was probed as loading control in all blots shown.