Impact of myeloid differentiation on apoptosis susceptibility. (A) Differentiation of P39 cells. Cells were stimulated for the indicated period with ATRA or vitamin D₃ in the absence or presence of z-VAD-fmk, followed by immunocytofluorometric detection of the differentiation marker CD11b. (B-C) Differentiation-associated cell death. Cells treated as in panel A were analyzed for any of 3 apoptotic parameters, namely ΔΨ_m loss (B), phosphatidyl serine (PS) exposure (C), and PI staining (B-C). (D-G) Effect of NF-κB inhibitors on differentiating P39 cells. Cells were cultured with the indicated combinations of ATRA, vitamin D₃, BAY11-7082, and/or bortezomib for 24 hours (D-E) or 72 hours (F-G), then labeled with DiOC₆(3) plus PI (D,F) or FITC-labeled annexin-V plus PI (E,G), and subjected to flow cytometric analyses. This experiment was repeated 4 times, yielding comparable results. Values are expressed as means ± SD; n = 3.