Involvement of CD47 and αvβ3 integrin receptors in mediating cell death induced by the C-terminal domain of TSP-1. (A) NB4-LR1 cells were treated with ATRA (1 μM) for 2 days without or with the T3C1 recombinant fragment (3 μM) and then fixed by the addition of 2% PFA. (i) Cells cultured without T3C1 were successively incubated with mouse monoclonal antibody to CD47 (20 μg/mL) and polyclonal antiserum to the αv integrin subunit (1:50). (ii, iii) ATRA-treated cells cultured with the T3C1 fragment were successively incubated with rabbit antiserum to TSP-1 (R1; 1:50) and mouse monoclonal antibody to αv or CD47 (20 μg/mL). Alexa Fluor 594-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-mouse IgG were used as secondary antibodies. Vectashield containing DAPI for nucleus staining was used as mounting medium for confocal microscopy analysis. Yellow color in the merge confocal images indicates the superimposition of the 2 fluorochromes. (B, C) NB4-LR1 cells, untreated or treated with ATRA (1 μM), were incubated for 2 hours with 4N1-1 (25 to 200 μM), GRGDS (0.1 to 1 mM), 4N1-1 (100 μM), and GRGDS (100 μM) or the VTCG (1 mM) peptide control and then cultured for 4 days (B) or 2 days (C) with the T3C1 recombinant fragment (3 μM). (B) At 4 days of the treatment, cell death was quantified by annexin V labeling and expressed as percent of positive cell detection upon flow cytometry analysis. Results are expressed as mean ± SEM of 3 experiments. (C) At 2 days of the treatment, cell death induction was monitored by annexin V (top), DiOC6(3) (middle), or DCFH-DA (bottom) staining for detection of PS membrane outside exposure, mitochondrial transmembrane potential alteration, and ROS production, respectively. Results are expressed as mean ± SEM of 2 experiments.
