Extended Data Figure 4: Genetic and pharmacological manipulation of AMD1 in prostate cancer cells.

a–c, DU145 cells carrying doxycycline-inducible shRNA against AMD1 (sh3) were transduced with empty (Mock), sh3-resistant wild type (AMD1^sh3R), or processing-deficient (AMD1^sh3R/S229A) AMD1 constructs. AMD1 protein (a, representative experiment out of four), dcSAM abundance (b), and cell number expression (c) in the aforementioned cells (n = 4 independent experiments as indicated by dots). Asterisks indicate significant differences compared with the corresponding DU145 cells in the absence of doxycycline, and hash symbol indicates significant differences in the indicated comparison. Mean ± s.e.m. d, Effect of MTA (25 μM) on AMD1 silencing (sh3)-elicited anti-proliferative activity. MTA was administered at day 0 and cells were analysed at day 3 (n = 3 independent experiments as indicated by dots). e, Mtap gene expression levels in Pten^pc+/+ and Pten^pc−/− mice at the indicated time points (see Extended Data Fig. 1a) (3 months, n = 3 mice; 6 months, n = 6 mice). Median ± interquartile range. f, MTAP gene expression analysis in publicly available data sets (see Methods; N, normal; 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). g, h, Effect of pharmacological AMD1 inhibition with SAM486A on cell number (g and left h; DU145, n = 5; PC3, n = 4 independent experiments as indicated by dots), and anchorage-independent growth (right h, n = 3 independent experiments as indicated by dots) in PC3 or DU145 cells as indicated. Mean ± s.e.m. i, Effect of pharmacological AMD1 inhibition with SAM486A in established DU145 xenograft tumour volume (vehicle, n = 11 tumours; SAM486A, n = 10 tumours). Box-and-whisker plot. j, k, Effect of pharmacological AMD1 inhibition with SAM486A in activated T CD4 cell number (96 h (j), n = 3 independent experiments as indicated by dots) or IL-2 production (k, n = 3–6 independent experiments as indicated by dots). */^#P < 0.05; **/^##P < 0.01; ***P < 0.001. Dashed line indicates cell number (g, h left) or IL-2 abundance (j, k) in vehicle-treated cells. Student’s t-test (b–d, f–h, j, k) and one-tailed Mann–Whitney test (e, i).