Extended Data Figure 9: Contribution of proAMD1 phosphorylation downstream mTORC1 to the stability of the enzyme.

a, b, Representative western blot depicting the stability of wild type and S298A phospho-mutant Myc-AMD1-HA constructs in DU145 cells upon cycloheximide treatment (densitometry of proAMD1 and AMD1 is represented in right panels (a) and half-life reduction in proAMD1 (b) (n = 3 independent experiments). Mean ± s.e.m. c, Representative western blot depicting the stability of S298A phospho-mutant Myc-AMD1-HA construct in DU145 cells challenged with vehicle (V) or MG132 (5 μM) upon CHX treatment (densitometry of proAMD1 is represented in right panel, n = 3 independent experiments). Mean ± s.e.m. d, mTORC1 kinase activity (by means of RAPTOR immunoprecipitation from HEK293 cells) on GST–proAMD1^S229A or GST–proAMD1^S229A/S298A, using bacteria-purified proteins. GST–p70S6K is presented as positive control. AZD8055 is used as control of mTORC1 inhibition. e, Correlation analysis between p70S6K^pT389 and AMD1 densitometry values in prostate cancer specimens from Fig. 4c (n = 15 patient specimens). f, Quantification of AMD1 immunoreactivity in response to everolimus in tumour biopsies, on the basis of the ΔH score (n = 14 specimen pairs). g, Box-and-whisker plot of the immunoreactivity of mTOR downstream effectors (AKT^S473, RpS6^pS240/244, 4EBP1/2^pT70, eIF4G^pS1108) in cancer patients with (ΔH score for KI67 < 0; n = 6 specimen pairs) or without (ΔH score for KI67 ≥ 0; n = 4 specimen pairs) anti-proliferative tumour response upon treatment with everolimus. h, Schematic representation of the main findings of this study. Orn, ornithine; Put, putrescine; Spm, spermine; ODC1, ornithine decarboxylase 1; PIP₂, phosphatidyl inositol bisphosphate; PIP₃, phosphatidyl inositol triphosphate; *P < 0.05; **P < 0.01; NS, not significant. Arrows indicate specific immunoreactive/autoradiographic bands. One-tailed Student’s t-test (a–c), two-tailed Mann–Whitney test (g), and Spearman’s correlation analysis (e).