vpiRNA production in Aag2 cells.

<p><b>(A)</b> Northern blot of highly abundant vpiRNAs. Two individual DNA oligonucleotide probes (left panels), or a combination of these probes (right panel) were used to detect the small RNAs. The combination of probes was used in all subsequent small RNA blots. <b>(B)</b> Upper panel: Small RNA northern blot of vpiRNAs in the indicated cell lines after infection with DENV2. Lower panel: RT-PCR for DENV genomic RNA in the same samples used for the northern blot. Numbers on top indicate DENV genomic RNA levels (relative to Aag2 cells) as determined by RT-qPCR (n = 1). <b>(C)</b> Northern blot of DENV2 piRNAs in Aag2 cells infected with the DENV2 NGC or 16681 strain, both at an MOI of 0.5. <b>(D)</b> Northern blot of DENV2 piRNAs and <i>Aedes</i> miR-2940-3p in uninfected or DENV2 infected Aag2 cells. Where indicated, total RNA was subjected to β-elimination. <b>(E)</b> RT-qPCR for the indicated PIWI proteins after gene-specific knockdown (KD) in Aag2 cells normalized to a control KD (dsLuc). Bars represent the mean of three experiments +/- SEM. Statistical significance was determined using two tailed, unpaired student t-test. * p<0.05; **p<0.01. <b>(F)</b> Upper panel: Small RNA northern blot of vpiRNAs upon KD of the indicated PIWI proteins. RNA samples analyzed in E were pooled for this blot. Lower panel: Quantification of two independent blots including the one shown in the upper panel using ImageJ software. For the other blot, see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004452#pntd.0004452.s007" target="_blank">S1 Dataset</a>. Ethidium bromide staining of ribosomal RNA was used as loading control in panel A, B, C, and F.