Effect of 17-AAG on cultured cells isolated from a MMTV-NEU-NT solid tumour. Cells treated for 24hr at 37°C with vehicle (control) or 1.12μM 17-AAG. A) Western blots of NEU/HER2, B-RAF, C-RAF, HSP72, CDK4 and cyclin D1 in vehicle treated (lanes 1,3,5) and 17-AAG treated (lanes 2,4,6) cells. Intensity of bands were quantitated relative to GAPDH, (n = 5). B)31P MR spectra of cell extracts from control cells (bottom), or cells following 17-AAG treatment (top). Peak assignments are: phosphoethanolamine (PE), phosphocholine (PC), inorganic phosphate (Pi), glycerophosphoethanolamine (GPE), glycerophosphocholine (GPC), phosphocreatine (PCr) and nucleotide triphosphate (α-, β-, γ-NTP). Results were confirmed in six separate experiments, and representative spectra are shown. C) Percentage changes (relative to control) in cell number, phosphoethanolamine (PE), phosphocholine (PC), glycerophosphoethanolamine (GPE), glycerophosphocholine (GPC), phosphocreatine (PCr) and nucleotide triphosphate (β-NTP), phosphomonoesters (PME = PE + PC), PC/GPC, phosphodiesters (PDE = GPE + GPC) and PDE/βNTP levels in MMTV-NEU-NT tumour cells following treatment with 17-AAG (1.12 μM) for 24h. Results are presented as the mean ± SEM (error bars) of six separate experiments. *p ≤ 0.05, **p < 0.01, two-tailed unpaired t test was used for all comparisons.