Fig. 4

Halofuginone and amino acid starvation both induce AAR yet only the former still allows mTOR-dependent protein synthesis. a, b WRO cells were exposed for 4 and 8 h to 100 nM halofuginone (HF), 35 μM cycloheximide (CHX) or amino acid/serum growth factors deprivation (EBSS). Thereafter, the cell homogenates were assessed for markers of autophagy (LC3B, p62) (panel a), of AAR (P-eIF2α, phosphorylated in Ser 51) (panel b), and mTOR ability to promote protein synthesis (P-S6, phosphorylated in Ser 235/236, and P-4E-BP1, phosphorylated in Thr 37/46) (panel b). Filters were, then stripped and probed with antibodies for total eIF2α or S6 or 4E-BP1 or β-Tubulin, as indicated. Densitometry analysis of the protein bands for p62/Tubulin, LC3B-II/I and P-(rp)-S6/(rp)-S6 is shown. (c) WRO cells were treated with 100 nM HF, in standard medium supplemented or not with 2 mM proline (PRO), or with 100 nM rapamycin (Rap) for the time indicated. The expression of total and phosphorylated (Ser 473) Akt, total and phosphorylated (Thr 37/46) 4E-BP1, and total and phosphorylated (Ser 2448) mTOR were assessed by immunoblotting. Filters were stripped and probed with anti-β-Tubulin as loading control. d, e WRO cells were exposed for 4 and 8 h to 100 nM HF in presence or absence of 2 mM proline (PRO) (panel d) or incubated for 8 h in EBSS (panel e) and the presence of puromycin incorporated in neosynthesized proteins was revealed by immunoblotting of cell homogenates. Filters were stripped and probed with anti-β-Actin as loading control. Blots shown in this Figure are representative of three independent experiments with reproducible data