Effect of 4EBP1 knockdown on PUMA expression and dual inhibitor-induced apoptosis. (A) Immunoblot of Jurkat cells transfected with different control (lanes 1 and 2) or 4EBP1 knockdown constructs (lanes 3 and 4). (B) qRT-PCR for BIM and PUMA mRNA in control or 4EBP1 knockdown cells after 48-hour treatment with diluent or 10 µM OSI-027. In panel B, *P < .001 compared with diluent control. (C-D) Immunoblot of cells transfected with empty vector or 4EBP1 shRNA and treated for 48 hours with (C) OSI-027 or (D) MLN0128. (E-F) Cells were stained with PI and subjected to flow microfluorimetry after treatment with (E) OSI-027 or (F) MLN0128. (G-H) Cells expressing control plasmid or 4EBP1 shRNA were transfected with pSPN empty vector or plasmid expressing shRNA-resistant S peptide-tagged 4EBP1, incubated for 48 hours (G) with or (H) without OSI-027, and harvested for annexin V (G) binding or (H) blotting. In panels E-G, *P < .05 relative to A8 control.