Figure 5.

5-FU induces RIP1-dependent necrosis in caspase-3 KO cells. C3KO or derived cells were treated with 5-FU (50 μg/ml). (a) Stable expression of shRIP1 or dominant-negative RIP1 (Flag-RIP1-DN) in C3KO1 cells was confirmed by western blotting. (b) Levels of HMGB1 release (medium) at 48 h with or without RIP1 inhibition. Necrostatin-1 (Nec-1, 15 μM). (c) Levels of HMGB1 release (medium) at 48 h with inhibitors of RIP1 (Nec-1) or MLKL (necrosulfonamide, NSA, 2 μM) in two C3KO lines. (d) Fractions of PI+/Annexin- C3KO1 cells at 48 h without or with Nec-1, NSA, ShRIP1, RIP1-DN. (e) Knockdown of caspase-8 in C3KO1 cells by siRNA confirmed by western blotting. (f) Levels of HMGB1 (medium) from C3KO1 cells with or without caspase-8 siRNA 48 h after 5-FU treatment. (g) Fractions of PI+/Annexin V-cells as treated in (c). (d and g) Data are the mean + S.E.M. of triplicate wells. **P<0.01