Pan-AKIs sensitize MM cells to TRAIL by inhibiting the expression of TRAIL-induced NF-κB target genes. (A) HMCLs were treated with MK-0457 for 3 hours and then incubated with TRAIL (2.4 ng/mL in RPMI 8226 and OPM-2, 9.6 ng/mL in RPMI 8226/R5 and U266, 300 ng/mL in JJN3) for 24 hours, after which endogenous A1/Bfl-1 and Mcl-1 were revealed by immunoblotting analysis. Blots were subsequently reprobed for actin expression to ensure equivalent loading and transfer of protein. Histograms represent the mean value ± SD of the ratio of A1/Bfl-1 or Mcl-1/actin normalized to the untreated control from blots of 3 independent experiments (*P < .001; Tukey-Kramer test). (B) Transfection of A1/Bfl-1 or Mcl-1, but not the unrelated nonspecific control siRNA (CONT), led to a decrease in A1/Bfl-1 or Mcl-1 protein expression in HMCLs without affecting the levels of the unrelated protein actin (insets). At 30 minutes after siRNA transfection, the cells were treated with TRAIL at the indicated doses (ng/mL) for the indicated time. Apoptosis was measured by annexin-V staining. Values are mean ± SD of 3 independent experiments (*P < .005; **P < .001 vs non-specific control siRNA [CONT]; Dunnet test). (C) RPMI 8226/R5 cells were infected with an empty lentiviral vector or lentivirus expressing A1/Bfl-1 or Mcl-1. All lentviral expression vectors coexpressed red fluorescent protein to monitor the infection by flow cytometry. Plot represents comparable red fluorescent protein expression of RPMI 8226/R5s infected with the parental retroviral vector or those encoding for Mcl-1 or A1/Bfl1. Whole-cell lysates of uninfected or virus-infected RPMI 8226/R5 cells were prepared and analyzed by western blot to confirm the overexpression of A1/Bfl-1 or Mcl-1 (inset). Pools of RPMI 8226/R5 were expanded after infection and treated with PHA-680632 (0.8 μM) and then incubated with TRAIL at the indicated doses for 24 hours. Cell death was measured by annexin-V staining. Values are mean ± SD of 3 independent experiments (*P < .005, vs PHA/TRAIL empty vector; Dunnet test). (D) HMCLs were treated as described in A and endogenous cIAP1, cIAP2, XIAP, and actin proteins were revealed by immunoblotting analysis. Bands were subjected to densitometric scanning, and histograms represent the mean value ± SD of the ratio of cIPA1, cIAP2, or XIAP/actin normalized to the untreated control from blots of 3 independent experiments (*P < .01; **P < .001; Tukey-Kramer test).