Figure 4

Inhibitory effects of spermidine on RTK signalling. A. PDGF-induced PDGFRβ phosphorylation is inhibited by spermidine in a TCPTP-dependent manner. Serum-starved TCPTP wt (+/+) and knockout (-/-) MEFs were incubated for 1 h in the presence or absence of spermidine and stimulated with PDGF as indicated. Cell lysates were resolved on SDS-PAGE and probed for phosphorylation at PDGFRβ pY1021. TCPTP (3E2) and tubulin blots serve as controls for TCPTP expression in wt and KO cells, and loading, respectively. B, C. Quantitation of PDGFRβ pY1021 phosphorylation levels normalized against tubulin in TC45 wt and KO cells (mean ± SEM; n = 2; *, P ≤ 0.05) B. or control- and 10 μM spermidine-treated cells (mean ± SEM; n = 3; *, P ≤ 0.05) C. following 15 minute PDGF treatment are shown. D. Spermidine attenuates EGF-induced EGFR signalling. Serum-starved HeLa cells were treated with 10 μM spermidine for 1 h, or left untreated, and stimulated with or without 50 ng/ml EGF for 5 or 15 minutes. Cell lysates were resolved on SDS-PAGE and immunoblotted with phospho-specific EGFR antibody. Tubulin was used as the loading control. A representative experiment out of three with similar results is shown. E. Quantitation of EGFR pY1068 phosphorylation levels normalized against tubulin in 10 μM spermidine treated and control EGF-stimulated HeLa cells (mean ± SEM; n = 3; *, P ≤ 0.05).