(A) Drosophila D-Mel2 cells were treated with staurosporine (STS; 50 nM), actinomycin D (Act D; 600 nM), etoposide (10 µM) cycloheximide (CHX; 25 µM), or left untreated. The percentage of cells undergoing apoptosis in each culture was determined by direct morphological assessment after 18 h. (B) D-Mel2 cells were treated with actinomycin D (600 nM), cycloheximide (25 µM) daunorubicin, (20 µM), or left untreated, and phase contrast images were taken at 18 h. (C) D-Mel2 cells treated for 18 h with etoposide (10 µM), cycloheximide (25 µM) or actinomycin D (600 nM), or left untreated, were analysed by flow cytometry to determine the degree of cell death. The percentage of viable cells in each culture is indicated on each dot plot. (D) Drosophila D-Mel2 cells were treated either with actinomycin D (600 nM), cycloheximide (25 µM), etoposide (10 µM), or daunorubicin (20 µM), or left untreated. Cell lysates were prepared from treated cells after 18 h and levels of DEVD-AFC hydrolysis activity measured by the addition of lysate samples to reactions containing 50 µM DEVD-AFC, followed by measurement of the release of free AFC by fluorimetry. (E) Drosophila D-Mel2 cells were either left untreated, or were treated with actinomycin D (600 nM) in the presence or absence of z-VAD-fmk (20 µM). After 18 h, percentages of viable cells were measured by flow cytometry. Results are representative of three independent experiments.

(A) Drosophila D-Mel2 cells were either left untreated, or were incubated with actinomycin D (600 nM) in the presence or absence of z-VAD-fmk (20 µM). At the indicated times, lysates were prepared and analysed by Western blotting with antibodies directed against substrate and control proteins. (B) Lysates prepared from D-Mel2 cells treated with actinomycin D (100 nM) for 18 h were incubated with ³⁵S-methionine-labelled bicaudal, DIAP1, stubarista and Dronc. Samples were taken at the indicated times and analysed by SDS-PAGE/fluorography. (C) ³⁵S-methionine-labeled bicaudal was incubated either with actinomycin D-treated D-Mel2 cell lysate as above, or with recombinant DCP-1 and drICE, and samples taken at the indicated times followed by SDS-PAGE/fluorography analysis. (D) Peptide coverage of the caspase-cleaved bicaudal protein sequence obtained by mass spectrometry is underlined. (E) Individual aspartic acid residues in the bicaudal sequence, D139, D140 and D141 were converted by site-directed mutagenesis to alanine (aa139 and aa141) or glutamic acid (aa140) residues. ³⁵S-methionine-labelled wild type and mutant proteins were incubated in buffer or with recombinant drICE for 120 min, before analysis by SDS-PAGE and fluorography. (F) ³⁵S-methionine-labeled wild type and D141A mutant bicaudal proteins were incubated in actinomycin D-treated cell lysate as above and samples taken at the indicated times for analysis by SDS-PAGE/fluorography.

(A) Alignment of the Drosophila bicaudal and human βNAC amino acid sequences. (B) Human Jurkat cell-free extracts were treated with 50 µg/ml cytochrome c and 1 mM dATP, or 100 nM granzyme B, to activate caspases, or left untreated. ³⁵S-methionine-labelled βNAC was added and samples taken after 0, 30, 60 and 120 minutes for analysis by SDS-PAGE and fluorography. (C) Human Jurkat cell-free extracts were treated with 100 nM granzyme B with or without 10 µM z-VAD-fmk, or left untreated. ³⁵S-methionine-labelled βNAC was added and samples taken as above. (D) Residue D175 in βNAC was mutated to alanine by site-directed mutagenesis and ³⁵S-methionine-labelled wild-type and mutant βNAC added to cytochrome c/dATP-treated or untreated cell-free extracts. Samples were then taken and analysed as above.

(A) Drosophila D-Mel2 cells were either or left untreated, or were treated with actinomycin D (600 nM) in the presence or absence of z-VAD-fmk (20 µM). After 7 h, cell lysates were prepared and 500 µg of protein from each reaction was analysed by two-dimensional SDS-PAGE followed by silver-staining. Representative 2D gels are shown with caspase-induced alterations annotated. (B) Enlarged regions of silver-stained two-dimensional SDS-PAGE gels from control or actinomycin D-treated cell lysates are shown. Proteins reproducibly altered upon caspase activation are indicated. Protein identifications were obtained by MALDI-TOF mass spectrometry. Results are representative of at least three independent experiments. Blue squares represent protein spots that disappear, in a caspase-dependent manner, upon induction of apoptosis. Red circles represent new protein spots that appear in apoptotic cells. Spot numbers on the gel in Figure 2B refer to the protein numbers in Table 1.

(A) Drosophila D-Mel2 cells were treated with dsRNAs against diap1, dronc or bicaudal for 48 or 72 h and levels of each mRNA was then determined by RT-PCR analysis. (B) D-Mel2 cells were treated with the indicated dsRNAs and lysates prepared after 48 h. Samples were subjected to SDS-PAGE and Western blotting with antibodies specific for bicaudal or actin. (C) D-Mel2 cells were treated with the indicated dsRNAs for 48 h followed by addition of actinomycin D (600 nM). Levels of cell death in drug-treated versus untreated cultures were then assessed. Results shown are representative of three independent experiments ±SEM. (D) D-Mel2 cells were treated with indicated dsRNAs and the number of cells present in each culture after 0, 1, 2, 3 and 6 days was assessed by haemocytometer counts. Results shown are the mean of triplicate counts ±SEM, from a representative experiment. (E) D-Mel2 cells were treated with dsRNAs targeted against the indicated gene products and phase contrast images of cultures were taken after 3 days. Results are representative of at least three independent experiments.

(A) MCF7 cells were transfected with pcDNA3-βNAC and treated with siRNA oligos against βNAC, or p84 as a control. Lysates were prepared after 72 h and samples probed with antibodies against βNAC, p84 and XIAP. (B) MCF7 cells were treated with three siRNA oligos against βNAC and with an siRNA oligo against p84. After 48 and 72 h, the percentage of cell death in each culture was assessed. (C) MRC5 cells were treated with siRNA oligos and cell death levels assessed as described above. (D) HEK293T cells were treated with siRNA oligos against βNAC, p84 and casp-9 for 48 h and cell viability and cell death in each culture was assessed. (E) HEK293T cells were treated with siRNA oligos against βNAC or CASP-9 for 48 h and phase contrast images taken.