Fig. 2.
Usp27x expression sensitizes 293FT cells to apoptosis through a contribution from an autocrine TNF loop that involves caspase-8 activation when stimulated with PMA. A, A TNF neutralizing antibody (TNFab) blocks Dox + PMA induced apoptosis in 293FT overexpressing mUsp27xS cells while exogenous TNF enhances apoptosis. Cells were seeded and treated the next day for 24 h or 8 h as indicated. TNFab was added 30 min before stimulation with Dox + PMA (n = 4). B, Same cells transiently transfected with a decoy receptor for TNF that contains only the ectodomain of TNFR2 (GPI-TNFR2, control: GFP-N1) show reduced apoptosis when treated with Dox + PMA (24 h, n = 3). C, Caspase-8 deficiency blocks apoptosis. 293FT-TetR-3xFlag-mUsp27xS or two different polyclonal derivatives where the caspase-8 locus had been targeted by CRISPR/Cas9 (control: non targeting gEGFP-1, the knock-out efficiency is shown in the figure to the right), were treated as indicated for 24 h. Apoptosis was measured after 24 h of treatment by staining for active caspase-3, followed by flow cytometric analysis (n = 3). A–C, Data (means, ns: not significant (adjusted p value > 0.05)). Error bars represent SEM. *, **: significant, see 2 section