N-terminal truncation of KRS by caspase-8. (A) Starved HCT116 cells were treated for 24 h with the pan-caspase (Pan-cas) inhibitor Z-VAD-FMK, and secreted KRS was precipitated with TCA and detected by immunoblotting with anti-KRS antibody. (B) HCT116 cells were transfected with caspase-specific siRNAs, and their effect on KRS secretion was determined by immunoblotting with anti-KRS antibody. (C) The effect of a specific caspase-8 inhibitor, Z-VAD-IETD, on KRS secretion was determined by immunoblotting of isolated exosomes. (D) The effect of the caspase-8 inhibitor on the N-terminal truncation of KRS was determined using KRS containing two domains of Renilla luciferase (Rluc), one fused to each end. Firefly luciferase (Fluc) was used to monitor transfection efficiency. Error bars show means ± SD from a mean of three experiments. n = 3. **, P < 0.01. (E) The effect of starvation on the expression levels of different caspases in HCT116 cells was determined by immunoblotting with their specific antibodies. act., active. (F) Different amounts of caspase-8 were expressed in the starved HCT116 cells, and their dose-dependent effects on secretion of KRS were determined. (G) The effect of the caspase-8 inhibition on the starvation-induced interaction of endogenous KRS and syntenin was determined by coimmunoprecipitation of HCT116 cell lysates using anti-syntenin and anti-KRS antibodies. (H) The effect of the KRS D12A mutation on KRS interaction with syntenin was determined by coimmunoprecipitation. IP, immunoprecipitation. (I) The effect of caspase-8 inhibitor on the cellular interaction of the FLAG-KRS (WT, D12A)–Venus (N domain) and HA-syntenin–Venus (C domain) in starved HCT116 cells was monitored by a BiFC experiment as described in Fig. 2 B (60×).