Induction of NB4-LR1 cell death by the C-terminal domain of TSP-1. (A) Schematic representation of TSP-1 structural and functional domains. TSP-1 is a disulfide-bonded trimeric protein composed of 3 identical subunits with a molecular mass of about 140 kDa each. Each subunit comprises multiple domains: an amino-terminal domain, a procollagen homology region (PC), 3 types of repeated sequence motifs—designated type 1 (filled diamond), type 2 (open circle), and type 3 (filled square) repeat—and a carboxy-terminal domain. The recombinant fragments used in this study are identified by their amino-terminal and carboxy-terminal amino acids (aa). VTCG, RGDA, and RFYVVMWK peptide sequences identified from TSP-1 for its respective binding to CD36, β3 integrin, and CD47 membrane receptors are indicated. (B, C) NB4-LR1 cells untreated (broken line) or treated (solid line) with ATRA (1 μM) were cultured for 4 days in the absence or the presence of increasing doses (0.5 to 5 μM) of TSP-1 recombinant fragments corresponding to the N-terminal (NTSP-1; ▴), the type 1 repeat (3TSR; ▪), or the type 3 repeat/C-terminal domain (T3C1; •) of the molecule. Cell growth of NB4-LR1 cells was expressed as percent of untreated cells growth (B), and cell death, as quantified by annexin V labeling, was expressed as percent of positive cell detection upon flow cytometry analysis (C). Results are expressed as mean ± SEM of 3 experiments. (D) Nucleus morphology of NB4-LR1 cells untreated or treated with ATRA with or without the T3C1 TSP-1 recombinant fragment (3 μM) or the entire TSP-1 (0.075 μM), as analyzed by confocal microscopy using Vectashield containing DAPI as mounting medium.