Fig. 5: 4E-BP1 is involved in the regulation of cell survival following gemcitabine and TRAIL treatment

a, b MIA PaCa-2 cells expressing a small hairpin RNA (shRNA) directed against 4E-BP1 and control cells expressing a scrambled shRNA were seeded in 96-well plates at a cell seeding density of 3×10⁴ cells/cm². a The sensitivity of cells to gemcitabine and TRAIL combination treatment was assessed by MTT assay. Cells were treated with increasing amounts of gemcitabine (0.1–100 μM) for 24 h (n = 4) and/or 100 ng/ml TRAIL for 24 h (n = 4). All experiments were repeated three times and data are provided as means ± SEM. One representative experiment is shown. P-values were calculated using Student’s t-test to determine the statistical significance of the difference between cells expressing a scrambled shRNA and cells expressing a shRNA directed against 4E-BP1, both cell lines having been treated with 10 or 100 μM gemcitabine and 100 ng/ml TRAIL (*P < 0.05). b Lysates made from cells treated as in a were used to purify eIF4E using chromatography on m⁷GTP-Sepharose beads as described in Methods. The levels of eIF4E and of the 4E-BP1 associated with it were determined by SDS gel electrophoresis and immunoblotting. Total cell lysates were analysed in parallel. Quantification was carried out by densitometry using ImageJ and the ratios of 4E-BP1 to eIF4E in the m⁷GTP-purified samples (in arbitrary units) are indicated