PIM inhibition blocks cap-dependent translation in RS cells. (A) PIM depletion decreases activity of 4E-BP1 and S6. Cells were transfected with a nontargeting siRNA (control, Ctrl) or with a siRNA cocktail targeting all 3 PIM kinases (3×PIM). After 4 days, p-4EBP1 (S65) and p-S6 (S235/236) protein expression was assessed by immunoblotting. GAPDH served as a loading control. Densitometric quantifications of band intensities (p-4EBP1 and p-S6, normalized to 4EBP1 and S6, respectively) are indicated above the lanes. (B) SEL24-B489 downregulates PIM-specific 4EBP1 and S6 phosphorylation. Cells were incubated for 4 hours with DMSO alone or with increasing doses (0.1-10 µM) of SEL24-B489, harvested, and lysed. Expression of p-4EBP1 (S65) and p-S6 (S235/236) were analyzed by immunoblotting. GAPDH served as a loading control. Densitometric quantifications of band intensities are shown in supplemental Table 6. Data in A-B are representative of 3 independent experiments.