Role of MYC downregulation in PUMA expression. (A) Immunoblot of Jurkat cells treated with diluent (lane 1), 10 µM OSI-027 (lane 2), 1 µM MLN0128 (lane 3), or 10 nM rapamycin (lane 4) for 48 hours. (B) Forty-eight hours after transient transfection with empty vector or plasmid encoding the indicated 4EBP1 construct fused to S peptide, cells were harvested for immunoblotting. (C-E) Forty-eight hours after transient transfection with control shRNA or shRNAs targeting MYC, (C) PUMA mRNA, (D) protein, and (E) annexin V binding were assessed. In panel C, *P = .036 relative to control. In panel E, *P = .002 and **P = .008 relative to control. (F-G) Cells transfected with empty vector or MYC cDNA behind the constitutive CMV promoter were treated with diluent or 10 μM OSI-027 and harvested for (F) qRT-PCR or (G) immunoblotting. Results of drug-treated samples in panels C and F were normalized to each corresponding diluent-treated control, which was set to 1. In panel F, *P = .011 relative to control. In panels A,B,G, *nonspecific bands.