Extended Data Figure 5: PTEN–PI3K–mTORC1-dependent regulation of AMD1 in prostate cancer.

a, b, Amd1 protein quantification from Fig. 3a (a, n = 3 mice) and mRNA expression (b, n = 5 or 6 mice as indicated by dots) in Pten^pc−/− and Pten^pc+/+ mice of the indicated age. Median ± interquartile range. c, AMD1 gene expression analysis in publicly available data sets (see Methods; number of patients analysed: ref. 48, normal n = 29, prostate cancer n = 150; ref. 46, normal n = 12, prostate cancer n = 76; ref. 47, normal n = 9, prostate cancer n = 17). d, Representative western blot showing the expression of the indicated proteins upon doxycycline-inducible expression (24 h) of YFP–PTEN^WT or catalytically inactive YFP–PTEN^C124S in PTEN-deficient LNCaP prostate cancer cells (n = 3 independent experiments). e, Schematic representation of the PI3K signalling pathway and the pharmacological/genetic tools used in this study. f, ProAMD1 and AMD1 protein quantification from Fig. 3b (sample number as indicated by dots). g, AMD1 gene expression upon treatment (24 h) with vehicle (V, DMSO), rapamycin (R, 20nM) and Torin-1 (T, 250 nM for PC3 and DU145, 125 nM for LNCaP) (LNCaP, n = 8 for Torin-1 and n = 6 for rapamycin; PC3 and DU145, n = 6 independent experiments as indicated by dots). Mean ± s.e.m. h, Representative western blot analysis of AMD1 levels upon 24-h treatment of DU145 cells with vehicle, Torin-1 (mTORC1/2 inhibitor; 250 nM), PD032901 (ERK-MAPK inhibitor, PD; 100 nM), SP600125 (JNK-SAPK inhibitor, SP; 10 μM), and SB203580 (p38-MAPK inhibitor, SB; 5 μM) (n = 3 independent experiments). i, Impact of inducible RAPTOR silencing in DU145 cells on proAMD1 protein levels (doxycycline-induced, 250 ng ml⁻¹) (representative experiment out of n = 6). j, Impact of inducible TSC2 silencing in DU145 cells on proAMD1 protein levels (doxycycline-induced, 250 ng ml⁻¹) (representative experiment out of n = 6). *P < 0.05; **P < 0.01; ***P < 0.001. Red asterisk in western blots indicates non-specific band. Arrows indicate specific immunoreactive bands. Student’s t-test (c, f, g) and Mann–Whitney test (a, b).