Smac mimetic and dexamethasone cooperate to downregulate IAP protein levels. (A-B) Cells were treated for indicated times with 300 µM dexamethasone and/or BV6 (Jurkat: 7 µM, Reh: 0.3 µM) (A) or with 300 µM dexamethasone (B). Protein expression of cIAP1, cIAP2, and XIAP was analyzed by western blotting after short or long exposure (exp.) times of blots. (C-E) Cells were treated for 24 hours (Jurkat) or 72 hours (Reh) with 300 µM dexamethasone and/or BV6 (Jurkat: 7 µM, Reh: 0.3 µM) in the presence or absence of 10 µg/mL Enbrel (C), 1 µg/mL anti-TRAIL antibody (D), or 5 µg/mL anti-CD95 antibody (E); treatments with 1 ng/mL TNFα and BV6 (Jurkat: 7 µM, Reh: 0.3 µM) (C), 5 ng/mL TRAIL (D), or 40 ng/mL hexameric CD95 ligand (CD95L) were used as PCs to demonstrate the effectiveness of blocking antibodies. (F-K) Jurkat cells were transfected with nonsilencing control siRNA (siCtrl) or siRNA against TNFR1 (siTNFR#1, siTNFR#2), DR5 (siDR5#1, siDR5#2), or CD95 (siCD95#1). Expression of death receptors was analyzed by western blotting, and asterisk indicates unspecific bands (F,H,J). Cells were treated for 24 hours with 300 µM dexamethasone and/or 7 µM BV6; treatments with 1 ng/mL TNFα and 1 µM BV6 (G), 1 μg/mL DR5 agonistic antibody Lexatumumab (ETR2) (I), or 40 ng/mL hexameric CD95 ligand (CD95L) (K) were used as PCs to demonstrate the efficacy of gene silencing. Cell death was determined by forward/side scatter analysis and flow cytometry. Mean and SD of 3 experiments performed in triplicate are shown; **P < .01; n.s., not significant.