Fig. 3.

hUsp27x_L or mUsp27x_S overexpression sensitizes 1205Lu and WM1158 human melanoma cells to TNF- and pIC induced apoptosis.  A, Overexpression of enzymatic active mUsp27x_S or hUsp27x_L specifically sensitizes 1205Lu to TNF and pIC induced apoptosis. Wilde Type (WT), mUsp22, Usp27x_S/L-inducible (mUsp27x_S-C87A is catalytically inactive [20]) 1205Lu cells were treated 72 h with doxycycline (Dox) and/or 100 ng/ml TNF / 50 µg/ml poly-I:C (pIC). Active caspase-3 as a marker for apoptosis was determined by flow cytometry. Shown is the rate of active-caspase-3 positive cells (n = 5). B, Induction of mUsp27x_S or hUsp27x_L enhances apoptosis in TNF treated WM1158 cells in a dose dependent manner. WT or inducible WM1158 cells were pre-stimulated 24 h with doxycycline to induce Usp27x or Usp22 and afterwards indicated concentrations of TNF were added for additional 24 h. Active caspase-3 was determined by flow cytometry (WT, n = 7; TetR-GFP-mUsp27x_S, n = 8; TetR-GFP-hUsp27x_L, n = 5; TetR-GFP-mUsp22, n = 3). C, Induction of hUsp27x_L enhances apoptosis in pIC treated WM1158 cells in a dose dependent manner independently of the mitochondrial pathway. GFP-hUsp27x_L-inducible WM1158, Bax^−/−/Bak^−/− GFP-hUsp27x_L- inducible WM1158 or constitutional overexpressing Bcl-X_L GFP-hUsp27x_L-inducible WM1158 cells were pre-stimulated 48 h with Dox to induce Usp27x and afterwards indicated concentrations of pIC were added for 4 h. Again, active caspase-3 was determined by flow cytometry (TetR-GFP-hUsp27x_L treated with 50/ 100 ng/ml pIC, n = 3; other samples: n = 4). D, The mitochondrial pathway does not restrict caspase-8 processing or cFLIP_L decrease in hUsp27x_L overexpressing and pIC treated WM1158 cells. DISC-lysates of indicated doxycycline (Dox) inducible WM1158 cells. Cells were or were not pre-treated 48 h with Dox and then pIC was added to indicated samples for 4 h. Proteins were detected by Western blot. Arrow-heads indicate specific signal (n = 1). E, pIC-induced apoptotic cell death in Usp27x over-expressing WM1158 melanoma cells is not dependent on RIPK1 kinase activity. GFP-hUsp27x_L inducible WM1158 cells were pre-stimulated 48 h with Dox. Cells were then pre-treated 30 min with Nec1 [10 µM] to inhibit RIPK1 kinase activity. Afterwards 50 ng/ml pIC was added for 4 h. QVD [10 µM] was added as indicated. Active caspase-3 was determined by flow cytometry (n = 4). A-C, E, Data (means, ns: not significant (adjusted p value > 0.05)). Error bars represent SEM. *, **, ***: significant, adjusted p values, see 2 section