Caspase-independent cell death induced by TSP-1 in NB4-LR1 cells. NB4-LR1 cells untreated or treated with ATRA (1 μM) were cultured for 4 days with or without the NTSP-1, 3TSR, or T3C1 fragment (3 μM) or the entire purified TSP-1 (0.075 μM). NB4-LR1 cells were also treated with staurosporine (STP; 5 μM) for 3.5 hours. (A) Caspase-3-like activity was determined in cell extracts by means of a colorimetric assay of the DEVD-pNa substrate hydrolysis. NB4-LR1 cells treated with STP with or without the broad-spectrum caspase inhibitor z-VAD-fmk (25 μM) were used as positive control for the detection of caspase-3-like activity as measured by spectrophotometry. OD indicates optical density. (B) SDS cell lysates (about 10 μg proteins corresponding to 10⁵ cells) prepared from NB4-LR1 cells were processed for Western blotting for analysis, under reducing conditions, of caspase-3 or caspase-8 activation, through cleavage of their precursor forms, cleavage analysis of the caspase substrate PARP, or the antiapoptotic protein Bcl-2. Polyclonal antibodies specific to caspase-3 or caspase-8 (1:500), monoclonal antibodies specific to PARP (1:200), or Bcl-2 (1:1000), respectively, were used. (C) Genomic DNA from NB4-LR1 cells (10⁶) was extracted, as described in “Materials and methods,” and electrophoresed on 2% agarose gel and then analyzed upon ethidium bromide visualization.