Overexpressed c-IAP1 interferes with TPA-induced THP1 cell differentiation. (A) Flow cytometry analysis of CD11b membrane expression in THP1 cells treated with 20 nM TPA for indicated times (h) in the presence or absence of 100 nM leptomycin B (LMB). Gray histograms indicate treated cells; and white histograms, untreated cells. (B) Western blot analysis of GFP expression in nuclear (N) and cytoplasmic (C) extracts from THP1 cells transfected with wild-type (wt-c-IAP1) and LRM2-mutated (c-IAP1-LRM2^*) GFP-c-IAP1. (C) Flow cytometry analysis of CD11b expression in GFP-positive THP1 cells transfected with pEGFP empty vector (vector) or wild-type (wt-c-IAP1) or LRM2-mutated (c-IAP1-LRM2^*) GFP-c-IAP1 constructs. Cells were treated with 20 nM TPA for indicated times (h). Gray histograms indicate treated cells; and black lines, control untreated cells. (D) Fluorescence microscopy analysis of THP1 cells transfected with pEGFP empty vector (vector) or LRM2-mutated GFP-c-IAP1 construct (c-IAP1-LRM2^*). Cells were incubated with 20 nM TPA for 48 hours and labeled with an anti-CD11b Ab (red). Nuclei were stained with Hoechst 33342 (blue). Magnification × 600.