Figure 6: Enhanced PHD3 expression inhibits EGFR signalling.

(a) PHD3 loss attenuates the hypoxia-mediated inhibition of EGFR signalling. Immunoblot of G141 tumour cells expressing control or PHD3 shRNA, following exposure to 21% (−) or 1% O₂ (+) for 48 h±EGF (20 ng ml⁻¹) for 5 min. (b,c) EGFR phosphorylation and signalling is attenuated by PHD3. Immunoblot with (P-)EGFR (b) and (P-)ERK (c) of G55 cells expressing PHD3 or GFP control. (d,e) Internalization of EGFR assessed by immunofluorescence in G55 cells expressing PHD3 or GFP control non-stimulated or stimulated with EGF (20 ng ml⁻¹) for the indicated times. The co-localization of EGFR with EEA1 induced by EGF is significantly increased in cells expressing exogenous PHD3 in comparison with the controls. The increased internalization induced by PHD3 overexpression is blocked by dynasore (d). Quantification of the increase in EGFR/EEA1 co-localization relative to non-stimulated cells (n=50–200) is shown in e. (f) Overexpression of PHD3 enhances the recruitment of the endocytic adaptor Eps15 to EGFR. Immunoblot of Eps15 of immunoprecipitated EGFR (IP α-EGFR) from G55 cells expressing PHD3 or GFP control that were non-stimulated or EGF stimulated for the indicated times. Western blot images (a–c,f) have been cropped for presentation. Full-size images are presented in Supplementary Fig. 8. All values are means+s.e.m., Student’s t-test *P<0.05; **P<0.01; ***P<0.001. Scale bar, 10 μm.