Nature Structural & Molecular Biology: doi: /nsmb.3308

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2 Supplementary Figure 1 Analysis of CED-3 autoactivation and CED-4-induced CED-3 activation. (a) Repeat experiments of Fig. 1a. (b) Repeat experiments of Fig. 1b. (c) Quantitative analysis of three independent CED-3 autoactivation experiments shown in a and Fig. 1a and CED-4-induced CED-3 activation experiments shown in b and Fig. 1b. In each experiment, the combined intensity (Ia-t) of active CED-3 bands (17 kda and 15 kda bands very close together and indicated with red brackets) at the 25-min, 45-min, 60-min, or 90-min time point of each CED-3 protein (wild-type or mutant) and the intensity (Iz-25) of the CED-3 zymogen band at the 25-min time point were quantified using the Image J software (See Online Methods). The relative level of the CED-3 zymogen activation (Ca-t) at each time point (45-min, 60-min, or 90-min) in each experiments was determined as follow: Cat=(Ia-t-Ia-25)/Iz-25 (t indicates 45-min, 60-min, or 90-min time point). The average level of the CED-3 zymogen activation (Ca-t) at each time point was then determined from three independent experiments. Error bars are s.e.m. Compared with the corresponding Ca-t from wild-type CED-3, *** P < ** P < * P < 0.05 (two-sided t-tests). (d) Autoradiographs of SDS-PAGE showing time-course of activation of the CED-3 zymogen harboring a catalytic-site mutation, G360S, with or without oligomeric CED-4. Experiments were performed as described in Fig. 1.

3 Supplementary Figure 2 CED-3::GFP localization, purity of isolated C. elegans nuclei and CED-3 activity assays.

4 (a,b) The GFP localization pattern in C. elegans germ cells. Images of DIC, Hoechst, GFP and GFP/Hoechst merged from the exposed gonads of a wild-type hermaphrodite adult carrying a complex transgenic array containing the P hsp GFP constructs (a) and a nontransgenic wild-type hermaphrodite adult (b) are shown. The animals were imaged 3 hours after the heat-shock treatment. Scale bars indicate 10 μm. (c) Representative DIC and GFP images from the exposed gonad of a hermaphrodite adult animal carrying the P ced- 3ced-3::gfp integrated transgene are shown. The white arrowheads indicate live germ cells and the red arrowhead indicates an apoptotic germ cell. Scale bar indicates 10 μm. Insets show enlarged images of the apoptotic germ cell (upper) and a live germ cell indicated by the bottom white arrowhead. From nine dissected gonads of adult hermaphrodites, 13 out of 15 apoptotic germ cells show a similar diffuse CED-3::GFP pattern. (d) 5 μl of ccis4810 nuclei or 10 μl of ccis4810 worm lysate were resolved by 12% SDS-PAGE and detected by immunoblotting using antibodies to GFP (LMN-1::GFP, a nuclear membrane marker), α-tubulin (a cytoskeleton marker), DLG-1 (a plasma membrane marker), HSP-60 (a mitochondrial marker), and HDEL proteins (an ER marker), respectively. ccis4810 is an integrated transgene that expresses a nuclear lamin GFP fusion. Purified nuclei were without cytoskeletal and other membrane contaminations. (e,f) Autoradiographs of SDS-PAGE showing CED-3 activity assays. 10 ng of the active CED-3 protease were incubated with 35 S-Methionine-labeled CED-9 in the presence of 4 μl of C. elegans nuclei as in Fig. 3b, 4 μl of recombinant NPP- 14 (125 nm final concentration) as in Fig. 4d, or 4 μl of the caspase inhibitor, iodoacetic acid (10 nm final concentration). Cleavage of the 31 kda CED-9 protein by active CED-3 generated a 24 kda cleavage product.

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6 Supplementary Figure 3 Analysis of CED-3 autoactivation and CED-4-induced CED-3 activation in the presence of C. elegans nuclei. (a) Repeat experiments of Fig. 3a. (b) Repeat experiments of Fig. 3b. (c) Repeat experiments of Fig. 3c. (d) Quantitative analysis of three independent CED-3 activation experiments without or with nuclei (shown in a-c and Fig. 3a-c, respectively). In each experiment, the combined intensity (Ia-t) of active CED-3 bands (17 kda and 15 kda bands very close together and indicated with red brackets) at the 25-min, 45-min, 60-min, or 90-min time point of each CED-3 protein (wild-type or mutant) and the intensity (Iz-25) of the CED-3 zymogen band at the 25-min time point were quantified using the Image J software (see Online Methods). The relative level of the CED-3 zymogen activation (Ca-t) at each time point (45-min, 60-min, or 90-min) in each experiments was determined as follow: Cat=(Ia-t-Ia-25)/Iz-25 (t indicates 45-min, 60-min, or 90-min time point). The average level of the CED-3 zymogen activation (Ca-t) at each time point was then determined from three independent experiments. Error bars are s.e.m. Compared with the corresponding Ca-t from wild-type CED-3, *** P < ** P < * P < 0.05 (two-sided t-tests).

7 Supplementary Figure 4 NPP-14 interacts with the CED-3 zymogen and its prodomain but not with the catalytic region of CED-3. (a) Results of pull-down experiments between GST or GST-NPP-14 and the bacterial lysate containing 3.5 nm CED-3(A449V)-FLAG or CED-3( , A449V)-FLAG (see Online Methods). The A449V mutation in CED-3 was used to block self-activation of the CED-3 zymogens in bacteria so that their potential interaction with NPP-14 can be examined. The left panel shows Coomassie Blue staining. The middle and right panels show immunoblotting using an anti-flag antibody. (b) An uncropped gel image of the one shown in Fig. 4a (lanes 10-14). (c) Cell corpse assays in 3-fold transgenic embryos of the indicated genotypes after heat-shock treatment (see Online Methods). Data shown are mean ± s.e.m (n=15). ** P < 0.01 (two-sided t-tests)

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9 Supplementary Figure 5 Analysis of CED-3 autoactivation and CED-4-induced CED-3 activation in the presence of NPP-14. (a) Repeat experiments of Fig. 4c. (b) Repeat experiments of Fig. 4d. (c) Repeat experiments of Fig. 4e. (d) Quantitative analysis of three independent CED-3 activation experiments without or with NPP-14 (shown in a-c and Fig. 3c-e, respectively). In each experiment, the combined intensity (Ia-t) of active CED-3 bands (17 kda and 15 kda bands very close together and indicated with red brackets) at the 25-min, 45-min, 60-min, or 90-min time point of each CED-3 protein (wild-type or mutant) and the intensity (Iz-25) of the CED-3 zymogen band at the 25-min time point were quantified using the Image J software (see Online Methods). The relative level of the CED-3 zymogen activation (Ca-t) at each time point (45-min, 60-min, or 90-min) in each experiments was determined as follow: Cat=(Ia-t-Ia-25)/Iz-25 (t indicates 45-min, 60-min, or 90-min time point). The average level of the CED-3 zymogen activation (Ca-t) at each time point was then determined from three independent experiments. Error bars are s.e.m. Compared with the corresponding Ca-t from wild-type CED-3, *** P < ** P < * P < 0.05 (two-sided t-tests).

10 Supplementary Table 1. Extra cell assays in different ced-3 mutant animals with or without the npp- 14(sm160) mutation. Genotype No. of extra cells Range of extra cells N2 0.1 ± ced-1(e1735) 0.1 ± npp-14(sm160) ced-1(e1735) 0.1 ± ced-1(e1735); ced-3(n2433[g360s]) 12.2 ± npp-14(sm160) ced-1(e1735); ced-3(n2433[g360s]) 12.1 ± ced-1(e1735); ced-3(n718[g65r]) 12.6 ± npp-14(sm160) ced-1(e1735); ced-3(n718[g65r]) 10.4 ± 0.3 * 8-12 ced-1(e1735); ced-3(n1040[l27f]) 10.3 ± npp-14(sm160) ced-1(e1735); ced-3(n1040[l27f]) 7.9 ± ced-1(e1735); ced-3(n2439[l30f]) 12.0 ± npp-14(sm160) ced-1(e1735); ced-3(n2439[l30f]) 8.9 ± 0.3 # 7-11 ced-1(e1735); ced-3(n2449[r51h]) 0.1 ± npp-14(sm160) ced-1(e1735); ced-3(n2449[r51h]) 0.1 ± The number of extra undead cells was scored in the anterior pharynx of larval stage 4 (L4) hermaphrodites (see Online Methods). Data shown are mean ± s.e.m. (n=20 animals). Statistical significance values were determined by two-sided Student s t-tests. * P < 0.01, compared with ced-1(e1735); ced-3(n718[g65r]) animals. P < 0.01, compared with ced-1(e1735); ced-3(n1040[l27f]) animals. # P < 0.01, compared with ced-1(e1735); ced-3(n2439[l30f]) animals.

11 Supplementary Table 2. The P ced-3 ced-3::gfp integrated transgene fully rescues the cell death defect of the ced-3(n2433) mutant. Strain Cell corpses ced-1(e1735) 31.9±1.7 ced-1(e1735); ced-3(n2433[g360s]) 0±0 ced-1(e1735); ced-3(n2433[g360s]); P ced-3 ced-3::gfp 31.7±2.0 Cell corpses were scored in 4-fold stage embryos. Data are mean ± s.d. (n = 15 embryos).

12 Supplementary Table 3. Loss of npp-14 increases germ cell death in several ced-3 mutants with specific prodomain mutations. Genotype No. germ cell corpses Range of germ cell corpses ced-1(e1735) 40.3 ± npp-14(sm160) ced-1(e1735) 36.0 ± 1.1 * ced-1(e1735); ced-3(n2433[g360s]) 0 0 npp-14(sm160) ced-1(e1735); ced-3(n2433[g360s]) 0 0 ced-1(e1735); ced-3(n718[g65r]) 0 0 npp-14(sm160) ced-1(e1735); ced-3(n718[g65r]) 0 0 ced-1(e1735); ced-3(n1040[l27f]) 0.1 ± npp-14(sm160) ced-1(e1735); ced-3(n1040[l27f]) 0.8 ± 0.2 # 0-3 ced-1(e1735); ced-3(n2439[l30f]) 0.1 ± npp-14(sm160) ced-1(e1735); ced-3(n2439[l30f]) 0.9 ± ced-1(e1735); ced-3(n2449[r51h]) 14.7 ± npp-14(sm160) ced-1(e1735); ced-3(n2449[r51h]) 15.5 ± Germ cell corpses were scored in the gonads of hermaphrodite animals 48 hours post L4 (see Online Methods). Data shown are mean ± s.e.m. (n=15 animals). Statistical significance values were determined by two-sided Student s t-tests. # P < 0.01, compared with ced-1(e1735); ced-3(n1040[l27f]) animals. P < 0.01, compared with ced-1(e1735); ced-3(n2439[l30f]) animals. * P < 0.05, compared with ced-1(e1735) animals.

13 Supplementary Table 4. Loss of npp-14 causes greater increase of germ cell death in several ced-3 mutants with specific prodomain mutations after UV irradiation. Genotype No. of germ cell corpses Range of germ cell corpses - UV + UV - UV + UV ced-1(e1735) 21.2 ± 0.8 a 34.4 ± 1.4 a npp-14(sm160) ced-1(e1735) 18.5 ± 0.8 a,* 29.0 ± 1.0 a,* ced-1(e1735); ced-3(n2433[g360s]) 0 b 0 b 0 0 npp-14(sm160) ced-1(e1735); ced-3(n2433[g360s]) 0 b 0 b 0 0 ced-1(e1735); ced-3(n718[g65r]) 0 b 0 b 0 0 npp-14(sm160) ced-1(e1735); ced-3(n718[g65r]) 0 b 0 b 0 0 ced-1(e1735); ced-3(n1040[l27f]) 0 b 0.8 ± 0.5 b npp-14(sm160) ced-1(e1735); ced-3(n1040[l27f]) 0.8 ± 0.3 b,# 7.2 ± 2.0 b, # ced-1(e1735); ced-3(n2439[l30f]) 0 b 0.2 ± 0.2 b npp-14(sm160) ced-1(e1735); ced-3(n2439[l30f]) 0.5 ± 0.2 b, 2.0 ± 0.7 b, ced-1(e1735); ced-3(n2449[r51h]) 3.1 ± 0.4 a 13.9 ± 1.0 a npp-14(sm160) ced-1(e1735); ced-3(n2449[r51h]) 2.9 ± 0.4 a 13.4 ± 0.9 a Germ cell corpses were scored in the gonads of hermaphrodite animals after UV irradiation (see online Methods). a Adult animals (24 hours post L4) were treated with or without 100 Jm -2 ultraviolet (UV) and germ cell corpses were scored 6 hours later (see Online Methods). b Adult animals (24 hours post L4) were treated with or without 100 Jm -2 UV and germ cell corpses were scored 24 hours later. Data shown are mean ± s.e.m. (n =15 animals). Statistical significance values were determined by two-sided Student s t-tests. # P < 0.01, compared with ced-1(e1735); ced-3(n718[l27f]) animals. P < 0.05, compared with ced-1(e1735); ced-3(n1040[l30f]) animals. * P < 0.01, compared with ced-1(e1735).