Quantitative analysis of <i>in vivo</i> CAT expression from promoter/CAT constructs.

<p>(<b>A</b>) The midgut samples from synchronized 4^th instar larvae (30 larvae/sample) were taken at 24 and 72 hours post 3^rd molt. (<b>B</b>) The carcass samples were from the same larvae as the midgut sampling. CAT quantity in each sample was defined as CAT unit/min of the reaction/µg protein. Mean and standard deviation are shown (N = 5−6). * Indicating significantly different (p<0.05) from the −0.06 kb construct at the same developmental time point.</p

Effects of <i>THAP</i> and <i>ATF-2</i> expression knockdown on development and fertility.

<p>The siRNA vector is driven by the <i>Drosophila hsp70</i> short promoter (see M&M). Constitutive over expression is drive by the immediate early gene promoter (Vyazunova and Lan, 2010). In the co-transfected groups, two expression vectors were added at 1∶1 ratio. In the single expression vector groups, pBS plasmid was added in at 1∶1 ratio to normalized the total amount of expression vector each group received. Thirty larvae were synchronized on Day 1 2^nd instar; siRNA expression was induced via heat shocked at 37°C for 24 hours on Day 1 of 2^nd and 4^th instar, respectively. Heat shock-treatment was not applied to the EGFP and SCP-2EGFP groups. (<b>A</b>) Developmental progression and mortality. The same letters above the bars in each construct represent that the mean values did not differ from other constructs significantly (p>0.05) in paired t-tests within each observation. Lower case letter represents heat shock treated group, capital letter represents non-heat shocked group. (<b>B</b>) Female Fertility (after the 1^st bloodmeal). Fertility is defined as viable 2^nd instar larvae/female. Survived pupae in each group from (Fig. 6A) were separated by sex and adults emerged in separated cages. Adults from each group (8–12) were mated with 10 wild type opposite sex (WF = wild type female or WM = wild type male). Eggs of blood-fed females were hatch 5–6 days after egg deposition. The average fertility/female in each mating group is presented. Mean and standard deviation are shown (N = 3). The same letters above the bars in each construct represent that the mean values did not differ from other constructs significantly (p>0.05) in paired t-tests within each observation. Capital letters above the bar represent non-heat shock groups; lower case letters above the bar represent heat shock-treated groups.</p

<i>In vivo</i> AeSCP-2 transcription in the larval midgut under the influence of transfected expression vectors.

<p>The siRNA vector is driven by the <i>Drosophila hsp70</i> short promoter. Constitutive over expression is drive by the Baculovirus immediate early gene promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046948#pone.0046948-Vyazunova1" target="_blank">[17]</a>. In the co-transfected groups, two expression vectors were added at 1∶1 ratio. In the single expression vector groups, pBS vector plasmid was added in at 1∶1 ratio to normalized the total amount of expression vector each group received. Larvae were synchronized on Day 1 2^nd instar; siRNA expression was induced via heat shocked at 37°C for 24 hours on Day 1 of 2^nd and 4^th instar, respectively. Heat shock-treatment was not applied to the EGFP and SCP-2EGFP groups. Day 1 4^th instar larvae (10 larval midguts/sample) were taken after the 2^nd heat shock-treatment. Relative (vs. <i>rpL8</i>) AeSCP-2 mRNA levels from each sample were determined via RT-qPCR. Mean and standard deviation are shown (N = 3). The same letters above the bars in each construct represent that the mean values did not differ from other constructs significantly (p>0.05) in paired t-tests with the vector control.</p

Nucleotide sequences of primers for PCR and oligos for siRNA.

*<p>Capital letters represent sequences of perspective genes; lower case letters represent added sequence for cloning purpose or the loop in the hairpin structure.</p

Tissue-specific transcriptional activities of the −1.6 kb <i>AeSCP-2</i> construct and efficiency for driving siRNA expression in transfected larvae.

<p>(<b>A</b>) Expression knockdown of <i>AeSCP-2</i> via the −1.6 kb <i>AeSCP-2</i> promoter driven siRNA expression. Tissues were collected from the same larvae (10 larvae/sample). Mean and standard deviation are shown (N = 3). (<b>B</b>) Fifteen synchronized 24 hour-old 4^th instar larvae were randomly selected and the total RNA was extracted from each individual larva. Mean and standard deviation are shown (N = 15). Relative (vs. <i>Actin-2</i>) AeSCP-2 mRNA levels from each sample were determined via RT-qPCR. * Indicating significant difference (p<0.05) from that of the empty vector construct.</p

Effects of expression knockdown of the transcription factors (Table 1, 24 h) on the progression of development, mortality, and female fertility.

<p>The siRNA vector is driven by the <i>Drosophila hsp70</i> short promoter. Thirty larvae were synchronized on Day 1 2^nd instar, heat shock at 37°C started on Day 1 of 2^nd instar through pupal stage and adults were returned to 26°C. (<b>A</b>) Temporal/spatial transcription profiles of the transcription factors in 4^th instar larvae via semi-quantitative RT-PCR (30 cycles). (<b>B</b>) RNA sample of pooled 24 h 4^th instar larval midguts (10 larvae/sample) was taken from randomly selected larvae in each respective group (triplicate batches). Relative quantity of mRNA (vs. <i>rpL8</i>) was determined via RT-qPCR analysis. Mean ± standard deviation (N = 3). * Indicate significantly different (<i>p</i><0.05, paired t-test) from the vector control. (<b>C</b>) Developmental progress and mortality was recorded daily. The same letter as the vector control above the bars represents that the mean values did not differ from the control significantly in paired t-test (<i>p</i>>0.05). Different letters above each construct represent that the mean values were significantly different between constructs (<i>p</i><0.05). (<b>D</b>) Female fertility was measured as producing viable 2^nd instar larvae per blood-fed female (after the 1^st bloodmeal) in 3–6 batches (>5 female/batch) of each mating group. Each group in panel “A” had ≥5 surviving pairs per batch. Mean and standard deviation are shown (N = 3−6).</p

Schematic diagram showing THAP- and ATF-2-regulated AeSCP-2 expression in the midgut of feeding 4^th instar larvae.

<p>Arrows indicate up regulation, bar represents down regulation, “?” denotes unknown factors.</p

Nuclear proteins bound to the <i>AeSCP-2</i> −1.6/−1.3 kb regulatory sequence.

<p>Nuclear proteins bound to the <i>AeSCP-2</i> −1.6/−1.3 kb regulatory sequence.</p

Quantitative analysis of <i>in vivo</i> CAT expression from co-transfection of transcription factor siRNA vector and AeSCP-2 promoter/CAT constructs (1∶1 ratio).

<p>In the promoter/CAT group, <i>hsp70</i> short promoter vector plasmid was added in at 1∶1 ratio to normalize the total amount of promoter/CAT each group received. Larvae were synchronized on Day 1 2^nd instar, heat shocked at 37°C for 24 hours on Day 1 of 2^nd and 4^th instar, respectively. Day 1 4^th instar larval samples were taken after the 2^nd heat shock-treatment. (<b>A</b>) The midgut samples from 4^th instar larvae (30 larvae/sample) were taken at 24 hour post 3^rd molt. (<b>B</b>) The carcass samples were from the same larvae as the midgut sampling. (<b>C</b>) Larval midgut endogenous transcription of <i>AeSCP-2</i> in −1.3 kb/CAT and siRNA co-transfected larvae. Ten synchronized 24 h 4^th instar larvae were pooled from each batch of the co-transfection experiment (in panel A, −1.3 kb). Relative <i>AeSCP-2</i> mRNA levels (vs. <i>Actin-1</i>) from each sample were determined via RT-qPCR. Mean and standard deviation are shown (N = 3). * Indicating significantly different (<i>p</i><0.05) from the promoter/CAT control.</p

An increase in autophagy activity in AcMNPV-infected SL-HP cells in comparison with non-infected SL-HP cells under starvation pressure.

<p>(A) Distribution of GFP-HaAtg8 punctual spots in non-infected SL-HP cells starved for 4.5 h, (B) Distribution of GFP-HaAtg8 punctual spots in AcMNPV-infected SL-HP cells starved for 4.5 h at 2 h of post-infection, showing the increase of autophagosomes in comparison with starved SL-HP cells, (C) Assay of acid phosphatase (ACP) activity between non-infected and infected SL-HP cells starved for 6 h at 4 h of post-infection.</p