Figure 1.

Cell death induction by DCC requires caspase-9. Human embryonic kidney 293T cells were transiently transfected as described (13) with the pCMV control plasmid (cont.), the DCC expression plasmid pDCC-CMV-S (DCC), or the DCC expression plasmid and the netrin-1-encoding plasmid pGNET1myc (DCC + net.) in the presence of the p35 expression construct pBabe-p35 (A), or of the dominant negative caspase-3, -8, or -9 expression construct (B). Cell death was analyzed by using trypan blue as described in ref. 13. (C) Mock (cont.) or DCC (DCC) transfected 293T cells were incubated or not with 20 μM Zvad-fmk (Zvad), IETD-fmk (IETD), DEVD-fmk (DEVD), or LEHD-fmk (LEHD) 24 h after the beginning of the transfection. Cell death was then analyzed by using trypan blue (as in A and B) 24 h later. (D) Caspase-9 −/− cells were transfected with a mock plasmid (cont.), DCC-expressing plasmid (DCC), or the caspase-8-expressing plasmid pcDNA-casp.8 (casp.8). (E and F) IMR32 cells were cultured for 48 h in the presence of culture medium issued of 293-EBNA-netrin-1 cells producing extracellular netrin-1. After this conditioned growth, cells were either further cultured with netrin-1-containing medium (+ net.) or with medium devoid of netrin-1 (− net.). In the latter case, cells were incubated or not with 20 μM LEHD-fmk (+LEHD). Cell death was then measured either by trypan blue staining at the indicated time following netrin-1 withdrawal (E) or by TUNEL reactivity after 24 h of netrin-1 withdrawal (F).