Figure 3. Discovery and validation of PAF.

(A) The 25 kDa and 15 kDa fragments are produced by caspase-dependent cleavage of Par-4 at its D131 residue. Cells were treated with vehicle or TRAIL (100 ng/ml) in the presence or absence of zVAD-fmk (left panel), or transfected with the indicated expression constructs or vector control (right panel). Whole cell lysates were then subjected to Western blot analysis.

(B) Recombinant PAF induces apoptosis specifically in cancer cells. A panel of cancer or normal cell lines was treated with recombinant TRX, TRX-Par-4 or TRX-PAF (100 nM each) for 24 h and scored for apoptosis.

(C) PAF induced apoptosis is dependent on FADD and caspase 8. PC-3 cells expressing dnFADD or FLIP, or parent PC-3 cells were treated with recombinant TRX, TRX-Par-4 or TRX-PAF (100 nM each) for 24 h and scored for apoptosis.

(D) PAF induced apoptosis is independent of cell surface GRP78. Par-4-null MEF cells were transfected with vector, Par-4 or PAF and expression of Par-4 or PAF in the CM was confirmed by Western blot analysis (lower panel). The CM was preincubated with antibody (Ab) for Par-4 (NT), GRP78 or control IgG, then applied to A549TR and H460 cells for 24 h to score for apoptosis (upper panel).

(B–D) The cells were scored for apoptosis by ICC for active caspase 3. The apoptosis data represent mean ± SD from three independent experiments. Asterisk (*) indicates statistical significance (P < 0.001) based on Student’s t test.