Transient expression of AaCASPS16 induced apoptosis in C6/36 cells.

<p>Plasmids expressing C-terminally Flag-tagged AaCASPS16-WT, AaCASPS16-C300A and GFP were transfected into C6/36 cells separately. Caspase inhibitor z-VAD-FMK was added at 2 h before transfection of pIE-AaCASPS16, and proteasome inhibitor MG132 was added at 8 h before cells were harvested. Mock treated cells, GFP expressed C6/36 cells, and AaCASPS16-C300A expressed C6/36 cells were used as controls. At 24 h post transfection, cells were subjected to the following analyses: <b>(A)</b> Photographs of cells were taken under microscope (Scale bar indicated 50 μm). <b>(B)</b> Cell lysates were prepared and 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. <b>(C)</b> Cell lysates were subjected to immunoblotting analysis using antibody against Flag and β-tubulin. The data in (B) were presented with the SD from three independent experiments, and statistical significance was calculated by <i>t</i> test, ** <i>P</i> < 0.01. NS: not significant.</p

AaCASPS16 was processed in apoptosis triggered by UV and Act D treatment.

<p>C6/36 cells were treated with Act D (1.0 μg/mL) or UV treatment (200 μJ/cm²) separately and at 24 h post treatment, cells were subjected to the following analyses: <b>(A)</b> Cell pictures were taken under microscope (Scale bar indicated 50 μm). <b>(B)</b> Cell lysates were prepared and 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. <b>(C)</b> Cell lysates were analyzed by immunoblotting using antibody against AaCASPS16 and β-actin. 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. The data in (B) were presented with the SD from three independent experiments, and statistical significance was calculated by <i>t</i> test, **<i>P</i> < 0.01.</p

Expression profile of <i>Aacasps16</i> in developmental and adult stages.

<p>Total RNAs were prepared from 1st to 4th instar larvae, pupae, female and male adults and subjected to qRT-PCR analysis. The vertical axis represents the relative expression of <i>Aacasps16</i> in different developmental stages or different genders. Pupae samples were designated as the standard and set to 1. The data were presented with the SD from three independent experiments. ND: not detected.</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

AaCASPS16 underwent autocatalytic cleavage when expressed from <i>E</i>. <i>coli</i>.

<p>(A) C-terminally His-tagged AaCASPS16-WT (lane 3) and the putative catalytic site mutant C300A (lane 4) were expressed from <i>E</i>. <i>coli</i> and detected by immunoblotting using an antibody against the His-tag following SDS-PAGE. The migration of full-length AaCASPS16 and the cleaved subunits were indicated. BL21 only (lane 1) and the uninduced WT preparations (lane 2) were used as control. 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. (B) The schematic cartoon showing the molecular masses of the His-tagged full length AaCASPS16 with potential cleavage sites and the cleaved subunits.</p

Phosphorylation sites prediction in the prodomain of AaCASPS16.

<p>Phosphorylation sites in the AaCASPS16 prodomain were predicted at the GPS web server using the default parameters [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157846#pone.0157846.ref025" target="_blank">25</a>]. (A) The numbers and positions of the potential phosphorylation sites were indicated. (B) Distribution of different potential kinase groups was calculated.</p

Phylogenetic analyses of AaCASPS16 with selected insect caspases.

<p>The predicted amino acid sequence of AaCASPS16 was aligned with 17 selected insect caspases, and a phylogenetic tree was constructed in MEGA 5.0 using the neighbor-joining method. AaCASPS16 was indicated by black dot. Accession numbers in GenBank and VectorBase of sequences are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157846#pone.0157846.s003" target="_blank">S1 Table</a>.</p

DataSheet7.XLS

<p>Caprine parainfluenza virus type 3 (CPIV3) is a newly emerging pathogenic respiratory agent infecting both young and adult goats, and it was identified in eastern China in 2013. Cellular microRNAs (miRNAs) have been reported to be important modulators of the intricate virus-host interactions. In order to elucidate the role of miRNAs in madin-darby bovine kidney (MDBK) cells during CPIV3 infection. In this study, we performed high-throughput sequencing technology to analyze small RNA libraries in CPIV3-infected and mock-infected MDBK cells. The results showed that a total of 249 known and 152 novel candidate miRNAs were differentially expressed in MDBK cells after CPIV3 infection, and 22,981 and 22,572 target genes were predicted, respectively. In addition, RT-qPCR assay was used to further confirm the expression patterns of 13 of these differentially expressed miRNAs and their mRNA targets. Functional annotation analysis showed these up- and downregulated target genes were mainly involved in MAPK signaling pathway, Jak-STAT signaling pathway, Toll-like receptor signaling pathway, p53 signaling pathway, focal adhesion, NF-kappa B signaling pathway, and apoptosis, et al. To our knowledge, this is the first report of the comparative expression of miRNAs in MDBK cells after CPIV3 infection. Our finding provides information concerning miRNAs expression profile in response to CPIV3 infection, and offers clues for identifying potential candidates for antiviral therapies against CPIV3.</p

DataSheet12.XLS

<p>Caprine parainfluenza virus type 3 (CPIV3) is a newly emerging pathogenic respiratory agent infecting both young and adult goats, and it was identified in eastern China in 2013. Cellular microRNAs (miRNAs) have been reported to be important modulators of the intricate virus-host interactions. In order to elucidate the role of miRNAs in madin-darby bovine kidney (MDBK) cells during CPIV3 infection. In this study, we performed high-throughput sequencing technology to analyze small RNA libraries in CPIV3-infected and mock-infected MDBK cells. The results showed that a total of 249 known and 152 novel candidate miRNAs were differentially expressed in MDBK cells after CPIV3 infection, and 22,981 and 22,572 target genes were predicted, respectively. In addition, RT-qPCR assay was used to further confirm the expression patterns of 13 of these differentially expressed miRNAs and their mRNA targets. Functional annotation analysis showed these up- and downregulated target genes were mainly involved in MAPK signaling pathway, Jak-STAT signaling pathway, Toll-like receptor signaling pathway, p53 signaling pathway, focal adhesion, NF-kappa B signaling pathway, and apoptosis, et al. To our knowledge, this is the first report of the comparative expression of miRNAs in MDBK cells after CPIV3 infection. Our finding provides information concerning miRNAs expression profile in response to CPIV3 infection, and offers clues for identifying potential candidates for antiviral therapies against CPIV3.</p

DataSheet5.XLS

<p>Caprine parainfluenza virus type 3 (CPIV3) is a newly emerging pathogenic respiratory agent infecting both young and adult goats, and it was identified in eastern China in 2013. Cellular microRNAs (miRNAs) have been reported to be important modulators of the intricate virus-host interactions. In order to elucidate the role of miRNAs in madin-darby bovine kidney (MDBK) cells during CPIV3 infection. In this study, we performed high-throughput sequencing technology to analyze small RNA libraries in CPIV3-infected and mock-infected MDBK cells. The results showed that a total of 249 known and 152 novel candidate miRNAs were differentially expressed in MDBK cells after CPIV3 infection, and 22,981 and 22,572 target genes were predicted, respectively. In addition, RT-qPCR assay was used to further confirm the expression patterns of 13 of these differentially expressed miRNAs and their mRNA targets. Functional annotation analysis showed these up- and downregulated target genes were mainly involved in MAPK signaling pathway, Jak-STAT signaling pathway, Toll-like receptor signaling pathway, p53 signaling pathway, focal adhesion, NF-kappa B signaling pathway, and apoptosis, et al. To our knowledge, this is the first report of the comparative expression of miRNAs in MDBK cells after CPIV3 infection. Our finding provides information concerning miRNAs expression profile in response to CPIV3 infection, and offers clues for identifying potential candidates for antiviral therapies against CPIV3.</p