Fig 3. Degradation of macrophage cytoskeletal-associated proteins upon contact with Eh is mediated by caspase-6.

(a) THP-1 macrophages were pre-treated with increasing amounts of the pan-caspase inhibitor Z-VAD-fmk or DMSO as control, for 1 h prior to incubation with Eh (20:1 ratio) for 5 min. Cleavage of the indicated cytoskeletal proteins was assessed by Western blot. (b) THP-1 macrophages were pre-treated with 50μM Z-VAD-fmk, 50μM of the caspase-6 inhibitor Z-VEID-fmk or DMSO as a control, followed by incubation with Eh (20:1) for 5 min. (c) Immunoblot of caspase-6 and its lamin A/C in macrophages incubated with Eh (20:1) for the increasing incubation times. Dose-dependent inhibition of cleavage of the indicated cytoskeletal proteins (d) or caspase-6 and its substrate (e) by Z-VEID. To inhibit calpain activation in macrophages, cells were pretreated with E-64 prior to incubation with Eh (10:1) and cell lysis (f). Lysates were loaded onto SDS-PAGE and immunoblotted with the indicated antibodies. (g) THP-1 cells were transfected with 25 and 50nM of caspase-6 siRNA, or scramble siRNA. After 48 h, transfected cells were stimulated for 2 min with Eh, followed by Western blot with the indicated antibodies. (h) Direct cleavage of paxillin was assessed by incubated immunoprecipitated paxillin with recombinant caspase-6 (C-6), with or without Z-VEID. Immunoprecipitated lamin was used as a positive control.