Fig. 1: A phosphoproteomics-based discovery strategy reveals that inhibition of polyamine synthesis regulates the process of pre-mRNA splicing.

a, Liquid chromatography–mass spectrometry (LC–MS) analysis of ¹³C-labelled polyamine after inducible silencing of AMD1 (for 1 day (1d) or 2 days (2d)) with doxycycline (Dox). n = 4 independent biological replicates. b, Bi-dimensional density plot showing the effect of AMD1 silencing (2 days) on the phosphoproteome and the full proteome. FC, fold change. c, z-scored heat map of phosphorylation sites (p-sites) that are altered by AMD1 silencing (compared with control (no Dox)) clustered by k-means. d, Gene ontology (GO) enrichment of phosphoproteins from cluster 1 in c by biological process and cellular component. NCC, nucleobase-containing compound. e, z-scored heat map of percent spliced in (PSI) alternative splicing events that are altered by AMD1 silencing (shAMD1; 2 days versus control (no Dox)) in DU145 cells (Abs(ΔPSI) > 10%, P < 0.1). f, Validation of top alternative splicing events, shown as fraction of long (l) versus short (s) transcripts (l/s ratio) from gel densitometry. n = 3 or 4 independent biological replicates. g, Semi-quantitative PCR showing the effect of SAM486A (0.5 µM, 24 h) on alternative splicing of SAT1 and SMARCA1 and recovery after drug washout (24–72 h). n = 3 independent biological replicates. S, SAM486A; V, vehicle. h, SAT1 (left) and SMARCA1 (right) l/s ratio after SAM486A (48 h) treatment of tumoural and non-tumoural cells by gel densitometry. n = 3 independent biological replicates. Veh, vehicle. i,j, Principal components analysis (i) and z-scored PSI heat map (j) (Abs(ΔPSI) > 10%, P < 0.1) of exonic events in DU145 cells treated with shAMD1 and induced with doxycycline (100 ng ml⁻¹) and supplemented with polyamines (10 µM, 2 days). k,l, z-scored PSI heat map of alternative splicing exonic events (k; n = 5 independent mice) and gel densitometry of Sat1 and G3bp2 l/s ratios (l; vehicle: n = 5, SAM486A: n = 4 independent mice) after SAM486A (8 days, 10 mg kg⁻¹ day⁻¹) in skeletal muscle of wild-type mice (Abs(ΔPSI) > 10%, P < 0.1). m,n, Principal components analysis (m) and z-scored PSI heat map (n) of exonic events in Th-Mycn mice treated with DFMO (n = 5 independent mice) versus control (n = 4 independent mice). CD, control diet. One-tailed paired Student’s t-test (a); one-tailed one-sample t-test (f); two-tailed one-sample t-test (h); two-tailed Mann–Whitney U-test (l). Adjusted P value was obtained by the multiple testing correction (set counts and sizes (SCS) method) (d). Data are mean ± s.e.m. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05; NS, not significant (P ≥ 0.05).

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