Figure 6: PHD3 loss sustains proliferative signalling through EGFR.

(a) EGFR phosphorylation is enhanced following PHD3 loss. Immunoblot with two different phospho-specific antibodies against EGFR of G55 cells expressing PHD3 or scrambled control nonstimulated or stimulated with EGF (20 ng ml⁻¹) for the indicated times. (b,c) EGFR activity is required for the growth-promoting effect of PHD3 loss. G55 cells expressing control or PHD3 shRNA were cultured as spheroids in B27-supplemented serum-free medium±EGF (20 ng ml⁻¹). Where indicated, cells were pretreated with the EGFR inhibitor erlotinib (10, 20 μM) for 2 h before stimulation. The number of spheroids was quantified after 3 days (n=6; b). Nude mice transplanted with G55 cells expressing control or PHD3 shRNA were treated with vehicle or erlotinib and tumour growth was quantified (n=13–18; c). (d,e) PHD3 loss through deletion or promoter methylation occurs preferentially in glioblastomas without EGFR amplification. Analysis of CNA for EGFR and PHD3, as well as PHD3 promoter methylation (Meth) in glioblastomas from the TCGA cohort (n=501, tumours for which CNA data are available). High-level EGFR amplification is shown in red, PHD3 deletion in green and PHD3 promoter methylation in blue (d). PHD3 deletion or promoter methylation are significantly more common in tumours without EGFR amplification (e). Western blot images (a) have been cropped for presentation. Full size images are presented in Supplementary Fig. 11. All values are means+s.e.m., *P<0.05; **P<0.01; ***P<0.001.