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1.

Figure. From: Induction of apoptosis by Drosophila reaper, hid and grim through inhibition of IAP function.

Fig. 2. Schematic representation of the diap1 mutants. The DIAP1 protein is boxed, with the BIR domains indicated as cross-hatched regions and the RING domain in black. The gain-of-function mutants are indicated in bold and the loss-of-function mutants are in normal face. The figure is drawn to scale.

Lakshmi Goyal, et al. EMBO J. 2000 February 15;19(4):589-597.
2.

Figure. From: Induction of apoptosis by Drosophila reaper, hid and grim through inhibition of IAP function.

Fig. 7. diap1 functions downstream of reaper, hid and grim to inhibit caspase activation. The ‘double inhibition’ model presented here and by Wang et al. (1999) indicates that diap1 is required to keep the caspases in check. The binding of reaper, hid and grim to diap1 results in the release of inhibition and therefore the activation of caspases and apoptosis.

Lakshmi Goyal, et al. EMBO J. 2000 February 15;19(4):589-597.
3.

Figure. From: Induction of apoptosis by Drosophila reaper, hid and grim through inhibition of IAP function.

Fig. 1. Mutations in diap1 modify reaper- and hid-induced cell death. A gain-of-function mutation in diap1, th6–3s, suppresses the GMR-reaper (B) (GMR-rpr/th6–3s) and GMR-hid (E) (GMR-hid/th6–3s) induced eye phenotypes. Reduction of diap1 activity as in th11–3e enhances the eye phenotypes associated with GMR-rpr (C) (GMR-rpr/th11–3e) and GMR-hid (F) (GMR-hid/th11–3e). The unmodified phenotypes are shown in (A) (GMR-rpr/+) and (D) (GMR-hid/+). The wild-type eye (not shown) is similar to that in (B).

Lakshmi Goyal, et al. EMBO J. 2000 February 15;19(4):589-597.
4.

Figure. From: Induction of apoptosis by Drosophila reaper, hid and grim through inhibition of IAP function.

Fig. 6. Cell deaths in the diap1 mutant embryo display the ultrastructural features of apoptosis. (A) Electron micrograph of a stage 6 wild-type embryo. All nuclei have normal ‘healthy’ morphology, and no signs of condensed chromatin can be detected until ∼7 h AEL (stage 11) (Abrams et al., 1993). (BD) Homozygous th11–3e mutant embryos were selected by morphology 4 h after egg laying and processed for electron microscopy. Representative micrographs of electron-dense nuclei from th11–3e homozygous mutant embryos showing condensed chromatin characteristic of apoptosis. The scale bar as represented in (D) is 1 μm.

Lakshmi Goyal, et al. EMBO J. 2000 February 15;19(4):589-597.
5.

Figure. From: Induction of apoptosis by Drosophila reaper, hid and grim through inhibition of IAP function.

Fig. 4. Gain-of-function mutations in diap1 diminish binding to REAPER and HID. Equal amounts of 35S-labeled wild-type and mutant DIAP1 proteins (IVT input) were tested for association with GST, GST–REAPER and GST–HID in an in vitro binding assay. Wild-type DIAP1 protein shows specific binding to GST–REAPER and GST–HID. Mutant DIAP1 proteins encoded by th6–3s and th23–4s show significantly reduced binding of GST–REAPER and GST–HID as compared with wild-type DIAP1. In contrast, a protein corresponding to th4, a loss-of-function allele of diap1, retains essentially normal affinity for REAPER and HID. The lower band visible in most lanes is due to initiation of translation at the second methionine in DIAP1 during in vitro synthesis. The th5 mutant translates into a truncated protein due to the introduction of a premature stop codon. The IVT input represents 10% of the in vitro translated protein used in each pull-down reaction.

Lakshmi Goyal, et al. EMBO J. 2000 February 15;19(4):589-597.
6.

Figure. From: Induction of apoptosis by Drosophila reaper, hid and grim through inhibition of IAP function.

Fig. 5. diap1 function is essential to prevent apoptosis. Embryos were labeled using the TUNEL procedure (A, B and C) to detect apoptotic nuclei (White et al., 1994) or with Oligreen (D, E and F) to stain DNA. (A) In wild-type embryos, the first evidence of apoptosis is seen at stage 11; the arrows point to clusters of apoptotic cells. (B) A th11–3e embryo aged to a physiological age of 7 h (7 h AEL). In diap1 loss-of-function mutants, such as th11–3e and th5, essentially all nuclei of embryos are TUNEL positive 7–8 h AEL. (C) th11–3e Df(3L)H99 double mutant embryos. As with th11–3e embryos, essentially all nuclei of the early H99 th11–3e double mutant embryos are TUNEL positive. Therefore, induction of apoptosis in th11–3e embryos does not require the activity of reaper, hid and grim, indicating that diap1 functions downstream from these genes. (D) Stage 5 (cellular blastoderm) wild-type embryo. (E) th11–3e embryo aged to 7 h AEL. At this stage, mutant nuclei have condensed chromatin characteristic of apoptosis, and the embryo is organized abnormally. (F) th11–3e Df(3L)H99 double mutant embryos. The absence of reaper, hid and grim does not attenuate the th11–3e phenotype, since the phenotype of th11–3e and the double mutant embryo is virtually indistinguishable (compare E and F).

Lakshmi Goyal, et al. EMBO J. 2000 February 15;19(4):589-597.
7.

Figure. From: Induction of apoptosis by Drosophila reaper, hid and grim through inhibition of IAP function.

Fig. 3. diap1 mutant alleles maintain their phenotype when expressed in S2 cells. (A) The wild-type and mutants of diap1 were expressed in S2 cells under the control of a viral promoter with a FLAG epitope tag at the N-terminus of the protein. Protein expression levels were analyzed by immunoblotting equal amounts of protein extracts using an epitope-specific antibody. Epitope-tagged versions of wild-type and mutant proteins are expressed at comparable levels and are indicated with arrows. The th5 mutant is a truncated protein due to the introduction of a premature stop codon. (B) The wild-type and mutant diap1 constructs were co-transfected with plasmids for the expression of REAPER and HID in a ratio of 3:4. A β–galactosidase plasmid was also co-transfected as a marker. The numbers of viable cells with β–galactosidase activity were counted and expressed as a percentage of the number of viable blue cells in the wild-type diap1-transfected wells, to assess the ability of the mutants of diap1 to inhibit reaper- and hid-mediated apoptosis. Except for th4, all the mutants maintained the phenotypes observed in vivo with respect to reaper- and hid-induced eye ablation (see Table I and text).

Lakshmi Goyal, et al. EMBO J. 2000 February 15;19(4):589-597.

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