Absence of mitochondrial intermembrane protein release upon NB4-LR1 cell treatment with the C-terminal domain of TSP-1. NB4-LR1 cells were either treated with ATRA (1 μM) for 3 days without or with the T3C1 recombinant fragment (3 μM) or treated with staurosporine (STP; 5 μM) for 3.5 hours. (A) Western blot analysis, under reducing conditions, of total cell extract and cytosolic S100 fraction prepared from 5 × 10⁶ cells, as described in “Materials and methods,” for detection of cytochrome c, using monoclonal antibody (1:100), in the respective fraction each corresponding to 10⁵ cells. (B) Immunofluorescence analysis of mitochondria protein in NB4-LR1 cells. Upon fixation by 2% PFA and permeabilization with 0.1% saponin, as described in the “Materials and methods,” cells were incubated with antibodies to cytochrome c (1:200), AIF (1:500), Smac/DIABLO (1:150), Omi/HtrA2 (1:150), or endonuclease G (6 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.