PDBu effects on TRAIL-Rs downstream signaling. (A) Flow cytometric analysis of TRAIL-Rs expression on primary AML cells treated with PDBu or without PDBu (DMSO) 2 nM for 3 days. Specific fluorescence histograms (▩) are superimposed to negative controls (isotype-matched irrelevant mAbs; empty histograms). A representative of 6 independent experiments is shown. (B) Detection of endogenous full-length (inactive) caspase-8, FLIP_L, and caspase-3 proteins in AML cells by Western blot. The proteins were revealed by the specific antibodies. β-actin control was used for protein loading. AML cells were treated for 3 days with DMSO (control), PDBu (20 nM), or TRAIL (25 ng/mL), alone or in combination. While TRAIL alone slightly induces caspase-3, PDBu alone also decreases FLIP_L levels. However, the combination of PDBu and TRAIL greatly activate caspase-3 and caspase-8 (decrease of full-length, inactive form) at the same time, completely down-modulating FLIP_L. A representative of 3 independent experiments is shown.