Differential gene expression profiles in JH-deprived and methoprene-exposed fat bodies.

<p>(A) Number of up-regulated (red) and down-regulated (blue) gene transcripts in JH-deprived fat bodies further treated with methoprene for 24 h. Fold change ≥2 and <i>P</i><0.05 were used as the cutoff criteria. (B) Significantly enriched gene ontology terms associated with up-regulated genes. (C) Significantly enriched KEGG pathways (top 10) of up-regulated genes. (D) Validation of RNA-seq data by qRT-PCR for genes associated with DNA replication. Fold change was calculated as: mRNA levels in methoprene-treated females/mRNA levels in precocene-treated females.</p

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Effects of <i>Met</i> RNAi on DNA replication and vitellogenesis.

<p>(A) <i>Met</i> knockdown efficiency and <i>VgA</i> mRNA levels in the fat body of the dsGFP control (iGFP), <i>Met</i> RNAi (iMet), and iMet further treated with methoprene for 48 h (iMet+JHA). **, <i>P</i><0.01 and ***, <i>P</i><0.001; n.s, no significant difference; n = 4–6. (B) Fat body cell ploidy (a–f) and DNA synthesis (g–i) of iGFP, iMet and iMet+JHA. Blue, nuclei; green, F-actin; red, <i>de novo</i> DNA synthesis. Enlarged images (4×) of a–c are shown in d–f, respectively with the white bar, 20 µm and red bar, 5 µm. (C) Statistical analysis for the diameter of cell nuclei. **, <i>P</i><0.001; n = 80–100. (D) FACS analysis of DNA contents in fat body cells. (E) Representative phenotypes of ovaries and ovarioles after iMet, iMet+JHA <i>vs.</i> iGFP. (F) Statistical analysis for length*width index of primary oocytes. ***, <i>P</i><0.001; n = 30.</p

Effects of JH deprivation and JHA application on DNA replication and vitellogenesis.

<p>(A) Fat body cell ploidy (a–h) and DNA synthesis (i–l). PAE10, 10-day-old adult females; P10d, adult females treated with precocene for 10 d; PM24h and PM48h, precocene-treated adult females further treated with methoprene for 24 h and 48 h, respectively. Blue, nuclei; green, F-actin; red, <i>de novo</i> DNA synthesis. Enlarged images (4×) of a–d are shown in e–h, respectively. White bar, 20 µm; red bar, 5 µm. (B) Statistical analysis for the diameter of cell nuclei. **, <i>P</i><0.01; ***, <i>P</i><0.001; n = 80–100. (C) FACS analysis of DNA contents in fat body cells. (D) Relative mRNA levels of <i>VgA</i> in the fat body. ***, <i>P</i><0.001; n = 8. (E) Morphology of ovaries and ovarioles. Scale bars: ovary, 5 mm; primary oocyte, 0.5 mm. (F) Statistical analysis for length*width index of primary oocytes. **, <i>P</i><0.01; ***, <i>P</i><0.001; n = 30.</p

Responsiveness of <i>Mcm4</i> and <i>Mcm7</i> to JH and <i>Met</i> RNAi.

<p>(A) Relative mRNA levels of <i>Mcm4</i> and <i>Mcm7</i> in the fat body of adult females treated with precocene (P10d) and those further treated with methoprene for 6, 12, 24 and 48 h (PM6h, PM12h, PM24h and PM48h, respectively). mRNA levels in precocene-treated fat bodies were used as the calibrator. *, <i>P</i><0.05, **, <i>P</i><0.01 and ***, <i>P</i><0.001 compared to P10d; n = 4–6. (B) <i>Mcm4</i> and <i>Mcm7</i> mRNA abundance in fat bodies of female locusts from 0 to 10 days post adult eclosion (PAE). *, <i>P</i><0.05 and **, <i>P</i><0.01 compared to PAE0; n = 4–6. (C) Relative levels of <i>Mcm4</i> and <i>Mcm7</i> transcripts in fat bodies of dsGFP control (iGFP), <i>Met</i>-RNAi (iMet), and iMet further treated with methoprene for 48 h (iMet+JHA). *, <i>P</i><0.05; n = 10–12. (D) Alignment of DNA element sequences containing E-box and E-box-like motifs in the upstream promoter regions of locust <i>Mcm7</i> and <i>Mcm4</i> with experimentally tested Met-binding consensus sequences except that of <i>DmKr-h1</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004702#pgen.1004702-Kayukawa1" target="_blank">[7]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004702#pgen.1004702-Li1" target="_blank">[9]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004702#pgen.1004702-Zou1" target="_blank">[34]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004702#pgen.1004702-Cui1" target="_blank">[36]</a>. (E) EMSA using the Mcm4 probe listed in (D) with fat body nuclear extracts of iGFP and iMet (shown is a representative short exposure; longer exposures in some experiments showed additional two bands of less interest; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004702#s2" target="_blank">Results</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004702#pgen.1004702.s007" target="_blank">Fig. S7</a>). (F) EMSA using Mcm7 probe listed in (D) and with fat body nuclear extracts of iGFP and iMet. For both (E) and (F), arrows indicate the most likely specific bands. FP, free probe.</p

<i>Mcm4</i> and <i>Mcm7</i> transcription and the JH-receptor complex.

<p>(A) Upper panels: western blot (WB) showing the expression of Flag-Met^1-3108 and V5-SRC in S2 cells; lower panel: immunoprecipitation (IP) showing the interaction of Flag-Met^1-3108 and V5-SRC in the presence of 10 µM JH III or methoprene. α-Flag, Flag antibody; α-V5, V5 antibody. (B) Luciferase assays after co-transfection of pGL4.10/<i>Mcm4</i>^{−933 to −37} or pGL4.10/<i>Mcm7</i>^{−933 to −11} into S2 cells compared with the pAc5.1 empty vector, pAc5.1/Flag-Met^1-3108 (Met) or/and pAc5.1/V5-SRC (SRC), with or without 10 µM JH III or methoprene (JHA) treatment. (C) EMSA using the Mcm4 probe and S2 cell nuclear extracts with expressed Flag-Met and V5-SRC and treated with JH III (10 µM). The arrow indicates the specific complex. FP, free probe. (D) EMSA using the Mcm7 probe and S2 cell nuclear extracts with expressed Flag-Met and V5-SRC and treated with JH III (10 µM). The arrow indicates the specific complex. FP, free probe.</p