Fig. 4: BENSpm elicits metabolic shielding and prevents polyamine depletion-triggered ES cell differentiation.
From: Polyamine-dependent metabolic shielding regulates alternative splicing
a, Schematic of the BENSpm molecule. b, Mechanism of action of BENSpm: antagonizing natural polyamine functions while mimicking metabolic shielding owing to its cationic nature. c, Representative binding mode of BENSpm on SF3A3. BENSpm is shown as green sticks, phosphorylatable residues (Ser365, Ser367 and Ser369) are in yellow and glutamic acid residues are in red. d, Selected 1H-1H-TOCSY NMR spectra of the SF3A3 fragment without (black) or with increasing BENSpm (blue to red). e, Average chemical shift perturbations from spectra in d, fitted to a 1:1 binding model with dissociation constant (s.e.m.). f, Autoradiography showing 32P incorporation into SF3A3 and CK1 after addition of SF3A3 and CK1 with or without BENSpm. n = 3. g,h, Semi-quantitative PCR gels (g) and densitometry (h) showing l/s ratios after BENSpm (10 µM, 48 h (BEN)) treatment in vehicle conditions and with AMD1 and ODC1 inhibition (SAM486A 1 µM + DFMO 50 µM, 48 h (S + D)) for validated alternative splicing candidates. n = 4 i–k, Polyamine quantification by LC–MS (i; n = 3), Nanog-GFP expression by flow cytometry (j; n = 2) and z-scored PSI heat map of alternative splicing events (k) in mouse ES cells transduced with Nanog-GFP and treated with SAM486A (0.5 µM, 48 h). l,m, Nanog-GFP flow cytometry (l; n = 2) and RNA-seq (shown as z-scored PSI heat map, m) of ES cells with Nanog-GFP treated with or without SAM486A (0.5 µM, 48 h) and BENSpm (10 µM, 48 h). One-tailed one-sample t-test with 1 as hypothetical value (h); two-tailed paired Student’s t-test for metabolomics (i). # indicates P values from two-tailed paired t-test for S + D versus S + D + BENSpm. Data are mean ± s.e.m.
