PMC

Constitutive and TNFα-induced RIP1 ubiquitination is reduced by Smac mimetic exposure. Jurkat cells were pretreated with BV6 (1 µM, 2 hours) before stimulated for 5 minutes with 2 µg/ml Flag-tagged TNFα (A) or 50 ng/ml TNFα (B). RIP1 ubiquitination was analyzed by immunoprecipitating TNFR1 using anti-Flag antibody (A) or by immunoprecipitating RIP1 followed by Western blot analysis.

Lack of caspase activation in Smac mimetic- and TNFα-induced cell death in FADD- or caspase-8-deficient cells. FADD- or caspase-8-deficient and WT Jurkat cells were pretreated with BV6 (1 µM, 2 hours) before being stimulated with 1 ng/ml TNFα for indicated time points. Caspase activation was analyzed by Western blot. Cleavage fragments are indicated by arrows. β-Actin served as loading control. Asterisk indicates an unspecific band. A representative experiment of two is shown.

FADD-deficient cells remain resistant to CD95-induced cell death even in the presence of Smac mimetic. FADD-deficient, caspase-8-deficient, or caspase-8-deficient and Bcl-2-overexpressing Jurkat cells (A) or WT (B) Jurkat cells were pretreated with BV6 (1 µM, 2 hours, black bars) before stimulating for 72 hours with 0.4 ng/ml agonistic anti-CD95 (A) or indicated concentrations of agonistic anti-CD95 (B). Cell death was analyzed by PI staining. Data are the mean and SD of three independent experiments performed in triplicate.

Requirement of RIP1 for Smac mimetic- and TNFα-induced cell death in FADD- and caspase-8-deficient cells. FADD-deficient (A) and WT (B) caspase-8-deficient (C) or caspase-8-deficient and Bcl-2-overexpressing (D) Jurkat cells were pretreated with BV6 (1 µM, 2 hours) before being stimulated with 1 ng/ml TNFα for 24 hours in the presence or absence of 20 µM zVAD.fmk or 30 µM necrostatin 1. Treatment with 1 ng/ml agonistic anti-CD95 for 24 hours was used as a positive control for apoptosis. Cell death was analyzed by PI staining. Data are the mean and SD of three independent experiments performed in triplicate. *P < .05. **P < .001.

Lack of DNA fragmentation in Smac mimetic- and TNFα-induced cell death in FADD-deficient cells. (A) Nuclear morphology was assessed by diamidine phenylindole dihydrochloride staining and fluorescence microscopy. FADD-deficient cells (a–d): (a) DMSO, (b) treatment with 1 µM BV6 for 8 hours, (c) treatment with 1 ng/ml TNFα for 8 hours, and (d) pretreatment with 1 µM BV6 for 2 hours before 8 hours of stimulation with 1 ng/ml TNFα for 8 hours. WT cells (e–h): (e) DMSO, (f) treatment with 1 µM BV6 for 8 hours, (g) treatment with 1 ng/ml TNFα for 8 hours, and (h) pretreatment with 1 µM BV6 for 2 hours before 8 hours of stimulation with 1 ng/ml TNFα for 8 hours. Representative pictures are shown; magnification, x60. (B) FADD-deficient and WT Jurkat cells were pretreated with BV6 (1 µM, 2 hours) before being stimulated with 1 ng/ml TNFα for 4 hours. DNA fragmentation was analyzed by FACS analysis of DNA fragmentation of propidium iodide-stained nuclei. Quantitative analysis of three independent experiments performed in triplicate with mean and SD (upper panel) and representative histograms of flow cytometric analysis (lower panel) are shown.

Smac mimetic sensitizes FADD- or caspase-8-deficient cells for TNFα-induced cell death. (A) WT (white bars), FADD-deficient (light gray bars), caspase-8-deficient (dark gray bars), or caspase-8-deficient and Bcl-2-overexpressing (black bars) Jurkat cells were pretreated with BV6 (1 µM, 2 hours) before being stimulated with 1 ng/ml TNFα for 4 hours. (B) WT (closed symbols) or FADD-deficient (open symbols) Jurkat cells were pretreated with BV6 (1 µM, 2 hours) before being stimulated with 1 ng/ml TNFα for indicated times. (C) FADD-deficient (left panel) or WT (right panel) Jurkat cells were pretreated for 2 hours with indicated concentrations of BV6 (white bars indicate 0 µM; light gray bars, 0.1 µM; dark gray bars, 1 µM; black bars, 3 µM) before adding the indicated concentrations of TNFα for 24 hours. (D) Primary leukemic blasts from three different children with ALL before the onset of chemotherapy were treated with 100 ng/ml TNFα and/or 100 nM BV6 or dimethyl sulfoxide (DMSO). In A to C, cell death was analyzed by PI staining; and in D, by forward side scatter analysis. In A to C, data are the mean and SD of at least three independent experiments performed in triplicate. **P < .001 comparing cells treated with BV6 and TNFα compared with DMSO-treated cells. In D, data are the mean of one experiment performed in duplicate.