NB4-LR1 cell death induction by TSP-1. (A) NB4-LR1 cells untreated or treated with ATRA (1 μM) with or without TSP-1 (0.1 μM) were cultured in microwell culture plate for 4 days. Cell growth was quantified by cell counting in a Malassez hemocytometer. Results are expressed as mean ± SEM of 2 experiments. (B) Morphology of MGG-stained untreated or ATRA-treated cells after 4 days of culture in the absence or the presence of purified TSP-1 (0.1 μM) showed cells with no chromatin condensation but damaged plasma membrane when cultured with TSP-1 with or without ATRA (arrows). Cells were analyzed using an upright epifluorescence microscope (Leica DMR, Leica, Rueil-Malmaison, France) through a Plan Apo 63×/1.32 numerical aperture oil immersion objective lens (Leica), and images were captured with a color camera LEI-750D CE system and acquired using the Leica QWin V2.2 software. (C) Flow cytometry analysis of FITC-annexin V and PI-stained untreated or ATRA-treated (1 μM) NB4-LR1 cells cultured with or without TSP-1 (0.1 μM) for 3 days. Results, expressed as percent of positive cells for annexin V labeling, are as follows: at day 2: untreated 3%, TSP-1 10%, ATRA 8%, ATRA plus TSP-1 22%; and at day 3: untreated 6%, TSP-1 19%, ATRA 15%, ATRA plus TSP-1 42%. (D) NB4-LR1 cells untreated or treated with ATRA (1 μM) were cultured for 4 days with or without increasing concentrations of purified TSP-1 (0.025 to 0.1 μM). Cell growth was expressed as percent of growth compared with untreated cells (•), and cell death, as quantified by annexin V labeling (▴), was expressed as percent of positive cell detection upon flow cytometry analysis: broken line indicates untreated cells; solid line, ATRA-treated cells. Results are expressed as mean ± SEM of 3 experiments.