Fig. 4. Combination treatment targets the mTOR pathway and alters the phosphorylation of 4E-BP1 in PDAC cells.

BxPC-3, MIA PaCa-2 cells and PANC-1 cells were treated with 100 μΜ gemcitabine for 24 h and/or 100 ng/ml TRAIL for 4 h. A unit of 15 μg of total protein lysate was analysed using western blotting. a PANC-1 cell lysates were analysed with antibodies directed against total mTOR, mTOR Ser²⁴⁴⁸, Raptor, Rictor, total 4E-BP1, 4E-BP1 Ser⁶⁵ and GAPDH. b BxPC-3, MIA PaCA-2 and PANC-1 lysates were analysed to look at the effect on levels and phosphorylation of 4E-BP1 at residues Ser⁶⁵, Thr ^37/46 and Thr⁷⁰ as well as levels of eIF4E. GAPDH was used as a loading control. c The change in phosphorylation of 4E-BP1 at Ser⁶⁵ in PANC-1 cells following combination treatment using TRAIL treatment for either 4 or 6 h was assessed by western blotting. PVDF membranes were immunoblotted with antibodies directed against total 4E-BP1 and 4E-BP1 residue Ser⁶⁵. d The relative levels of phosphorylation of 4E-BP1 at Ser⁶⁵ were quantified by scanning densitometry using ImageJ and the data are shown on the histogram as % of the values for untreated cells. All experiments were repeated three times and data are provided as means ± SEM. P-values were calculated using Student’s t-test to determine the statistical significance of the difference between untreated cells and cells treated with either gemcitabine or gemcitabine plus TRAIL (*P < 0.05 and ***P < 0.001)