Induction of apoptotic morphology in cell extracts is not blocked by inhibition of the CAD-like nuclease. (A) Induction of nucleosomal ladders in added nuclei by S/M extract is sensitive to ICAD and DEVD-fmk (lanes 3–8). Induction of nucleosomal ladders in added nuclei by E/X extract is sensitive only to ICAD (lanes 9–14). (DEVD-fmk added: none, lanes 1–5 and 9–11; 100 μM, lanes 6–8 and 12–14. ICAD added: none, lanes 3, 6, 9, and 12; 160 ng, lanes 4, 7, 10, and 13; 320 ng, lanes 5, 8, 11, and 14.) (B) Both S/M and E/X extracts can induce apoptotic events under conditions where the CAD-like nuclease is inhibited, provided that they retain active caspases (panels 3–5 and 9–11). E/X extracts, which normally induce apoptosis in the absence of caspase activity (panel 12), are unable to do so if ICAD is added along with DEVD-fmk (panels 13 and 14). Panel numbers refer to the gel lanes in A. These images come from the same incubations shown in A. Time-lapse microscopy analysis reveals that the C-shaped nuclei in panels 3–5 and 9–11 are apoptotic nuclei in which the nuclear envelope has ruptured after collapse of the chromatin against the nuclear periphery. (C) E/X extracts can fully induce apoptotic morphology in the absence of CAD-like activity, but this requires ongoing caspase activity. In a different experiment from that shown in A and B, the number of nuclei per microliter of extract was reduced. This gives a stronger induction of apoptotic morphology (compare a with B, panel 9), even when caspases (b) or CAD are inhibited (c). Extracts produce a range of morphologies in added nuclei. As in the experiment of A and B, simultaneous inhibition of both CAD and caspases abolishes morphological apoptosis in the extracts (d). These images were selected from images taken at random.