Figure 1: Loss of PHD3 enhances EGFR signalling.
From: PHD3 regulates EGFR internalization and signalling in tumours
(a,b) PHD3 loss enhances EGFR phosphorylation (a) and signalling (b). Immunoblot of (P-)EGFR (a) and (P-)ERK (b) of G55 cells expressing control or PHD3 shRNA that were non-stimulated or EGF stimulated for the indicated times. (c) Enhanced EGFR signalling following PHD3 loss is HIF-1/2α independent. Immunoblot of (P-)EGFR and (P-)ERK of G55 cells expressing control or PHD3 shRNA in combination with control, HIF-1α or HIF-2α or double HIF-1α/2α shRNA that were non-stimulated or EGF stimulated for 5 min. (d) Immunoblot of (P-)EGFR and (P-)ERK of G55 cells expressing control or PHD3 shRNA that were transfected with wild-type PHD3, the hydroxylase mutant PHD3-H196A or empty vector control and were non-stimulated or EGF stimulated for 5 min. (e) EGFR mediates 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) and transfected with control or EGFR siRNA. The number of spheroids was quantified after 3 days (n=6). (f) MAPK signalling is required for the growth-promoting effect of PHD3 loss. G55 tumour cells expressing PHD3 shRNA were cultured as spheroids in B27-supplemented serum-free medium without growth factors±treatment with the MAPK inhibitor U0126 (10 μM) and the number of spheroids was quantified after 3 days (n=6). (g) Constitutively active MAPK signalling phenocopies PHD3 deficiency. G55 cells expressing control or PHD3 shRNA were transfected with a constitutively active MAPK construct (MEK1-ERK) or control vector, cultured as spheroids in B27-supplemented serum-free medium±EGF/FGF and the number of spheroids was quantified after 3 days (n=6). Western blot images (a–d) have been cropped for presentation. Full-size images are presented in Supplementary Fig. 5. All values are means+s.e.m., Student’s t-test ***P<0.001.
