SfDredd underwent autocatalytic cleavage when expressed and purified from <i>E</i>. <i>coli</i>.

<p>C-terminally His-tagged <b>(A)</b> or N-terminally His tagged <b>(B)</b> SfDredd and their active site mutant SfDredd-C443A were expressed and purified from <i>E</i>. <i>coli</i> and detected by immunoblotting using antibody against His-tag following SDS-PAGE. Diagrams showing the full length SfDredd and cleaved subunits are on the right. A short vertical black line was used to indicate where lanes were removed and separate parts of the same Western blot image were joined together. His: His-tag, Pro: prodomain, LS: large subunit, L: linker, SS: small subunit.</p

Recombinant SfDronc possessed strongest activity on initiator caspase substrates.

<p>Wild type SfDronc and active site mutant SfDronc-C310A with C-terminal His-tag were incubated, respectively with 13 types of synthetic caspase substrates (20 μmol/L) and subjected to caspase activity assay. Caspase activity was indicated as the changes in relative fluorescence units (RFU) per minute. The data were presented with the SD from three independent experiments, and statistical significance was calculated by <i>t</i> test, ***<i>P</i> < 0.001, *<i>P</i> < 0.05.</p

Recombinant SfDredd possessed the strongest activity on effector caspase substrates.

<p>Wild type SfDredd and active site mutant SfDredd-C443A with C-terminal His-tag <b>(A)</b> or N-terminal His-tag <b>(B)</b> were incubated, respectively with 13 types of synthetic caspase substrates (20 μmol/L) and subjected to caspase activity assay. Caspase activity was indicated as the changes in relative fluorescence units (RFU) per minute. The data were presented with the SD from three independent experiments, and statistical significance was calculated by <i>t</i> test, ***<i>P</i> < 0.001.</p

The protein level of SfDredd increased in ActD-treated Sf9 cells.

<p>Sf9 cells were treated with ActD at a final concentration of 250 ng/mL. <b>(A)</b> Cell pictures were taken 6 h post ActD treatment (magnification ×200). <b>(B)</b> Sf9 cells were harvested 6 h after ActD (250 ng/mL) treatment, and cell lysates were analyzed by immunoblotting using antibody against SfDredd.</p

Phylogenetic analysis of SfDredd with selected insect caspases.

<p>The predicted amino acid sequence of SfDredd together with 27 selected insect caspases were aligned and the phylogenetic tree was constructed by MEGA 5.05 using the neighbor-joining method. The sequences include the following: SfDredd, SfDronc and Sf-caspase-1 from <i>Spodoptera frugiperda</i>, Spli-caspase-5 and Spli-caspase-6 from <i>Spodoptera litura</i>, Se-caspase-5 and Se-caspase-6 from <i>Spodoptera exigua</i>, Hv-caspase-6 from <i>Heliothis virescens</i>, Ha-caspase-5 and Ha-caspase-6 from <i>Helicoverpa armigera</i>, Ms-caspase-6 from <i>Manduca sexta</i>, Gm-caspase-6 from <i>Galleria mellonella</i>, Bm-caspase-1, Bm-caspase-3/a (BmICE), Bm-caspase-4, Bm-caspase-5 (BmDronc) and Bm-caspase-6 (BmDredd) from <i>Bombyx mori</i>, Ld-caspase-5 from <i>Lymantria dispar</i>, Pr-caspase-5 from <i>Pieris rapae</i>, AeDredd and AeDronc from <i>Aedes aegypti</i>, Dcp1, DECAY, DAMM, DrICE, STRICA, DmDredd and DmDronc from <i>Drosophila melanogaster</i>. Genbank accession numbers of sequences are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151016#pone.0151016.s004" target="_blank">S4 Table</a>. *: Bm-caspase-4 belongs to Lep-caspase-4 which is a peculiar caspase, whether it belongs to initiator or effector caspase has not been decided [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151016#pone.0151016.ref024" target="_blank">24</a>].</p

The sequence of SfDredd.

<p>The predicted amino acid sequence of SfDredd is shown in alignment with Dredd homologs from <i>Bombyx mori</i> (BmDredd), <i>Aedes aegypti</i> (AeDredd) and <i>Drosophila melanogaster</i> (DmDredd-PE). The amino acid residues identical among 4 Dredds are indicated by white letters within black boxes, the amino acid residues identical among 3 Dredds are indicated by white letters within dark gray boxes, and the amino acid residues identical between 2 Dredds are indicated by black letters within light gray boxes. The alignment was performed using ClustalX 2.1 and modified by GeneDoc 3.2. Secondary structures were predicted using JPred3. Box: catalytic center, closed circles: conserved residues responsible for the catalytic reaction, black arrow: predicted cleavage sites.</p

Knockdown of <i>SfDredd</i> led to decreased apoptosis induced by ActD.

<p><b>(A)</b> 5×10⁵ Sf9 cells were transfected with dsRNA (1 μg) of <i>SfDredd</i>-dsRNA or <i>GFP</i>-dsRNA, 24 h later, cells were harvested and subjected to immunoblotting using antibody against SfDredd. <b>(B-D)</b> 5×10⁵ Sf9 cells were transfected with dsRNA (1 μg) of <i>SfDredd</i>-dsRNA or <i>GFP</i>-dsRNA, 24 h later, cells were treated with ActD at a final concentration of 250 ng/mL for 6 h and subjected to the following analyses: (B) Cell images were taken under a microscope (magnification ×200). (C) Cell lysates were incubated with Ac-DEVD-AFC and subjected to caspase activity assay. Caspase activity was indicated as the changes in relative fluorescence units (RFU) per minute. The data were presented with the SD from three independent experiments, and statistical significance was calculated by <i>t</i> test, ***<i>P</i> < 0.001. (D) The cleavage of Sf-caspase-1 was detected by immunoblotting using an antibody against Sf-caspase-1.</p

SfDredd directly cleaved Sf-caspase-1 <i>in vitro</i>.

<p><b>(A)</b> Sf-caspase-1-C178A (300 nmol/L) was incubated with 300 nmol/L of C-terminally His-tagged SfDredd, SfDredd-C443A or SfDronc at 37°C for 1 h in Na-Citrate buffer and then subjected to immunoblotting using an antibody against Sf-caspase-1. <b>(B)</b> Sf-caspase-1-C178A (300 nmol/L) was incubated with 300 nmol/L or 600 nmol/L of C-terminally His-tagged SfDredd or SfDredd-C443A at 37°C for 1 h in Na-Citrate buffer and then subjected to immunoblotting using an antibody against Sf-caspase-1.</p

HCV replication up-regulates DR4 and DR5.

<p>(A) The mRNA level of DR4, DR5, DcR1, and DcR2 in 9–13 and Huh7 cells was measured using real-time RT-PCR. (B) Huh7 and 9–13 cell lysates were subjected to western blot analyses using a rabbit polyclonal antibody against DR4 or DR5. (C) The DR4 reporter plasmid (DR4/−1156; 100 ng) or DR5 reporter plasmid (DR5/−1192; 100 ng) was co-transfected with the <i>Renilla</i> luciferase reporter plasmid (100 ng) into 9–13 or Huh7 cells cultured in a 24-well plate. After 2 days, the cells were harvested, and the luciferase activity was measured. (A and C) The data from the 9–13 cells were normalized to Huh7 cells to directly show the fold induction caused by HCV. The data are presented with the SD from three independent experiments, and statistical significance was calculated by <i>t</i> test, * indicates a <i>p</i> value less than 0.05.</p

The sequence of AaCASPS16.

<p>Predicted amino acid sequence of AaCASPS16 was shown in alignment with Strica homologs from <i>Aedes aegypti</i> (AeCASPS15, AeCASPS16, AeCASPS17 and AeCASPS21) and the caspases from <i>Drosophila melanogaster</i> (Dronc, Dredd, Drice, Decay, Dcp-1, Strica and Damm). The amino acid residues identical among 12 caspases are indicated by white letters within black boxes, the amino acid residues identical among 9 caspases are indicated by black letters within dark gray boxes, the amino acid residues identical among 6 caspases are indicated by black letters within medium gray boxes, and the amino acid residues identical among 3 caspases are indicated by black letters within light gray boxes. The alignment was performed using DNAMAN 7.0. Secondary structures were predicted using JPred3. Underline: catalytic center, black arrow: predicted cleavage site.</p