Figure 6
Rapid signalling cascades centred around Src kinase in androgen-treated LNCaP cells leads to PHB charge differentiation. (a and b) Kinexus protein array data from LNCaP cells grown in media with charcoal-stripped serum for 72 h and then treated with ethanol or R1881 (10 nM) for 4 h. (a) Phosphorylation changes on components of the cell cycle machinery at G1/S. (b) Phosphorylation events in intracellular cell signalling proteins. (c) Western blot of total Src and Src (phospho-Tyr418) from LNCaP cells grown in media with charcoal-stripped serum for 72 h and then treated with ethanol or R1881 (10 nM) for 4 h. Bar graph represents densitometry data. (d) Q-PCR analysis of androgen-driven PSA expression in LNCaP cells grown in media with charcoal-stripped serum for 72 h and then treated with ethanol or R1881 (10 nM) for 4 h with or without Src inhibitor. (e) 2D western blot of PHB from LNCaP cells grown in media with charcoal-stripped serum for 72 h and then treated with ethanol or R1881 (10 nM) for 4 h with or without Src inhibitor. Blot represents isoelectric focussing of pH 3–10 horizontal and protein size vertical (4–12% gradient gel). (f) Luciferase assays of COS-7 cells transfected with pSG5-PHB plasmid and MCM5 and 6 promoter reporters in the presence or absence of Src inhibitor. Transfections normalised to β-galactosidase. (g) Luciferase assay of LNCaP/PHBcDNA cells grown in full serum, transfected with MCM6 reporter and treated with either doxycycline and enzalutamide for 24 h. Data represent the average of three independent experiments and is normalised to untreated cells. (h) Western blot of AR, PHB and histone H3 associated with the chromatin of LNCaP cells grown in media with charcoal-stripped serum for 72 h and then treated with ethanol or R1881 (10 nM) for 4 h, with or without Src inhibitor. (i) Bar graph indicating the relative densitometry of chromatin-associated AR, PHB and from LNCaP cells grown in media with charcoal-stripped serum for 72 h and then treated with ethanol or R1881 (10 nM) for 4 h, with or without Src inhibitor. All data are the mean±s.d. of three independent experiments performed in triplicate. *P<0.05 and **P<0.01 (t-test analysis).
