tLivin-induced apoptosis and necrosis are partially mediated by activation of JNK.

<p>(<b>A</b>) Cells of MelA1 clones were treated with 10 µM SP600125 1 h prior to administration of 2.5 µg/ml dox (where indicated) and harvested after 36 h along with untreated cells (con). Cells were stained with PI and the percent of cell death was measured by flow cytometry. *p<0.05. (<b>B</b>) 293T cells were transiently transfected with the indicated plasmids and harvested 24 h post-transfection along with untransfected cells (con). Cells were stained with PI and the percent of cell death was measured by flow cytometry. (<b>C</b>) 293T cells were transiently transfected with the indicated plasmids, harvested 24 h post-transfection and analyzed by western blot for the protein levels of p-c-JUN, c-JUN, JIP-1, tLivin and GAPDH.</p

tLivin activates an alternative form of cell death when apoptosis is inhibited.

<p>Cells of MelA1 clones were treated with 75 µM zVAD-fmk 1 h prior to addition of 2.5 µg/ml dox or 30 µM cisplatin and harvested after 36 h along with untreated cells (con). (<b>A</b>) Cells were stained with PI and the percent of cell death was measured by flow cytometry. *p<0.05. (<b>B</b>) Cells from each sample were divided into three aliquots and analyzed for cleavage of caspase 3 by western blot and for the percent of PI positive cells and DNA content by flow cytometry.</p

tLivin activates the JNK pathway.

<p>(<b>A</b>) 293T cells were either transiently transfected with empty vector (ev), tBID- or tLivin-expressing vectors, treated with 30 µg/ml etoposide for 24 h (etop) or left untreated (con). Cells were harvested at the indicated time points post-transfection and the phosphorylation of JNK, ATF-2 and p38MAPK was analyzed by western blot. (<b>B</b>) Cells of MelA1 clones were harvested at the indicated time points following administration of 2.5 µg/ml dox or following 30 min treatment with 200 nM anisomycin (A), control cells (con) were left untreated. Phosphorylation of JNK and c-JUN was analyzed by western blot. (<b>C</b>) 293T cells were either transiently transfected with empty vector (ev), tLivin- or tLivinΔBIR-expressing vectors, treated with 200 nM anisomycin for 30 min (A) or left untreated (con). Cells were harvested at the indicated time points post-transfection and phosphorylation of JNK and c-JUN was analyzed by western blot.</p

tLivin induces apoptosis of MelA1 cells.

<p>Cells of MelA1 clones were treated with 2.5 µg/ml dox and harvested at the indicated time points. Con, untreated cells; sts, cells treated with 0.5 µM staurosporine for 10 h. (<b>A</b>) Cells were stained with PI and the percent of cell death was measured by flow cytometry (top). Cells were analyzed for tLivin and GAPDH protein levels by western blot (bottom). (<b>B</b>) Cleavage of caspase 3 and PARP was analyzed by western blot. (<b>C</b>) DNA content was analyzed by flow cytometry.</p

tLivin induces apoptosis of A549 cells.

<p>Cells of A549 clones were treated with 2.5 µg/ml dox and harvested at the indicated time points. Con, untreated cells; etop, cells treated with 30 µg/ml etoposide for 48 h. (<b>A</b>) Cells were stained with PI and the percent of cell death was measured by flow cytometry (top). Cells were analyzed for tLivin and GAPDH protein levels by western blot (bottom). (<b>B</b>) Cleavage of caspase 3 and PARP was analyzed by western blot. (<b>C</b>) DNA content was analyzed by flow cytometry. (<b>D</b>) Cells were treated with 50 µM zVAD-fmk 1 h prior to addition of dox, harvested after 48 h, stained with PI and the percent of cell death was measured by flow cytometry. *p<0.05. (<b>E</b>) Cells were treated with 50 µM zVAD-fmk 1 h prior to addition of dox and harvested after 48 h. Each sample was divided into three aliquots and analyzed for cleavage of caspase 3 by western blot and for the percent of PI positive cells and DNA content by flow cytometry.</p

tLivin induces caspase-independent necrotic cell death of 293T cells.

<p>293T cells were transfected with an empty vector (ev) or with vectors expressing either tBid, tLivin or tLivinΔBIR and harvested at the indicated time points post-transfection. Con, untransfected cells; (<b>A</b>) Cells were stained with propidium iodide (PI) and the percent of cell death was measured by flow cytometry. (<b>B</b>) Cells were analyzed by western blot for cleavage of caspases and PARP. (<b>C</b>) zVAD-fmk was added 1 h before transfection. Cells were harvested 24 h post-transfection and stained with PI. The percent of cell death was measured by flow cytometry. *p<0.05. (<b>D</b>) Cells were analyzed for DNA content by flow cytometry (top). Representative cell cycle histograms of tBID- and tLivin-transfected cells (bottom). (<b>E</b>) Cells were examined by transmission electron microscopy 18 h post-transfection with empty vector (I) or a tLivin-expressing vector (II, III). Cells were incubated with 10 mg/ml sodium azide for 4 h (IV) or 30 µg/ml etoposide for 24 h (V). Features of necrosis: cytoplasmic vacuolation (white arrow) (II, III, IV) and rounded mitochondria with disrupted internal structures (black arrows) (III, IV) are evident in cells transfected with tLivin and in cells treated with sodium azide. Apoptotic cells (treated with etoposide) exhibited blebbing of the plasma membrane (black arrow) (V). (<b>F</b>) Cells were analyzed for DNA content by flow cytometry. (<b>G</b>) zVAD-fmk at 20 µM was added 1 h before transfection and cells were harvested 24 h post-transfection. Each sample was divided into three aliquots and analyzed for cleavage of caspase 3 by western blot and for the percent of PI positive cells and DNA content by flow cytometry. ND, Not determined.</p