Immunoblot analysis of the subcellular location of ArSLK during development.

<p>Cell fractionation was performed and tubulin and H3 were used as markers of the cytoplasmic and nuclear fractions, respectively. (<b>A</b>) Dp, diapause cysts; Pd, post-diapause cysts; Np, nauplius larvae. (<b>B</b>) Nauplius-destined oocyte and embryonic developmental processes of <i>Artemia</i>. EO, early oocytes; LO, late oocytes; EE, early embryos; LE, late embryos.</p

Sequence analysis of ArSLK.

<p>(<b>A</b>) Alignment of the amino acid sequence of the N-terminal kinase domain of ArSLK with those of SLKs from other species. The Ste20 signature sequence is highlighted and conserved amino acid residues are indicated by asterisks. The black shaded areas represent identical amino acid residues. (<b>B</b>) Analysis of the coiled-coil region of the ArSLK. The probability of formation of a coiled-coil structure was calculated for each residue with a window of 28 amino acids using the ‘COILS’ program. (<b>C</b>) Phylogenetic analysis of the amino acid sequences of SLKs from multiple species. GenBank accession numbers of the sequences used in the amino acid alignment and phylogenetic analysis are as follows: <i>Artemia parthenogenetica</i>, KC818632; <i>Drosophila melanogaster</i>, NP_726441; <i>Aedes aegypti</i>, XP_001649074; <i>Daphnia pulex</i>, EFX76394; <i>Danio rerio</i>, NP_001139073; <i>Homo sapiens</i>, NP_055535; <i>Xenopus laevis</i>, NP_001079164. <i>Cluex quinquefasciatus</i>, XP_001864707; <i>Nasonia vitripennis</i>, XP_001603525; <i>Xenopus troplicalis</i>, NP_001072623; <i>Mus musculus</i>, NP_033315; <i>Schistosoma mansoni</i>, AAN72832; <i>Clonorchis sinensis</i>, GAA30009.</p

The relative amounts of <i>ArSLK</i> mRNA detected by real-time PCR.

<p>The mRNA amounts were normalized to those of <i>tubulin</i> mRNA. (<b>A</b>) The relative amounts of <i>ArSLK</i> mRNA in oocytes and embryos of ovoviviparous and oviparous <i>Artemia</i>. EO, early oocytes; LO, late oocytes; EE, early embryos; LE, late embryos; Naup, nauplius larvae; Dp, diapause cysts. (<b>B</b>) The relative amounts of <i>ArSLK</i> mRNA during the hatching process of <i>Artemia</i>. Dp, diapause cysts; Pd, post-diapause cysts. The times (h) represent the incubation times of the post-diapause embryos. (<b>A, B</b>) Data are represented as mean <u>+</u> SE of n = 3 independent repeats. Statistical analyses of the differences between expression levels in the two pathways were performed by two-tailed, paired Student's <i>t</i> tests. No significant differences (<i>P</i><0.05) were detected.</p

Immunoblot of amounts of ArSLK protein during development.

<p>Tubulin was measured as a loading control. The developmental stages are labelled as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092234#pone-0092234-g003" target="_blank">Figure 3</a>. (<b>A</b>) ArSLK protein in oocytes and embryos of ovoviviparous and oviparous pathways. (<b>B</b>) Amounts of ArSLK protein during development of post-diapause cysts.</p

Immunoblot analysis of the subcellular locations of ArSLK and p26 after exposure to 42°C heat shock (A), acidic pH stress (B), and UV irradiation (C).

<p>Tubulin and H3 were used as markers of the cytoplasmic and nuclear fractions, respectively. C, control group with no stress treatment; R, nauplius larvae after stress that underwent subsequent stress-free incubation for 7 h.</p

Nucleotide sequences and positions of primers used in polymerase chain reactions.

<p>F and R indicate the forward and reverse directions, respectively.</p

Acetylation of Chromatin-Associated Histone H3 Lysine 56 Inhibits the Development of Encysted <i>Artemia</i> Embryos

<div><p>Background</p><p>As a response to harsh environments, the crustacean <i>artemia</i> produces diapause gastrula embryos (cysts), in which cell division and embryonic development are totally arrested. This dormant state can last for very long periods but be terminated by specific environmental stimuli. Thus, <i>artemia</i> is an ideal model organism in which to study cell cycle arrest and embryonic development.</p> <p>Principal Finding</p><p>Our study focuses on the roles of H3K56ac in the arrest of cell cycle and development during <i>artemia</i> diapause formation and termination. We found that the level of H3K56ac on chromatin increased during diapause formation, and decreased upon diapause termination, remaining basal level throughout subsequent embryonic development. In both HeLa cells and <i>artemia</i>, blocking the deacetylation with nicotinamide, a histone deacetylase inhibitor, increased the level of H3K56ac on chromatin and induced an artificial cell cycle arrest. Furthermore, we found that this arrest of the cell cycle and development was induced by H3K56ac and dephosphorylation of the checkpoint protein, retinoblastoma protein.</p> <p>Conclusions/Significance</p><p>These results have revealed the dynamic change in H3K56ac on chromatin during <i>artemia</i> diapause formation and termination. Thus, our findings provide insight into the regulation of cell division during arrest of artemia embryonic development and provide further insight into the functions of H3K56ac.</p> </div

A working model for the regulation of <i>Artemia</i> diapause by H3K56ac.

<p>Green circles represent H3K56ac.</p

Cell cycle arrest in <i>Artemia</i> diapause embryos.

<p>1–3 and 1’–3’ represent three embryonic developmental stages in ovoviviparous and oviparous pathway, respectively. 1 and 1’, early embryo (two days after the eggs entering into the ovisac); 2 and 2’, late embryo (four days after the eggs entering into the ovisac); 3, nauplius; 3’, diapause embryo. (<b>A</b>) Morphology of <i>Artemia</i> adults with embryos in the ovisac. (<b>B</b>) BrdU incorporation assay (upper panel) and corresponding DAPI staining (lower panel) with embryos or nauplius of the two reproduction pathways. Black arrows indicated the representative positive signal. (<b>C</b>) Western blotting analysis of cell division-related molecules at each stage of ovoviviparous and oviparous reproduction pathways. Tubulin was used as a loading control for the whole protein extracts, and H3 used as a loading control for the total histones.</p

NM induces an artificially arrest via increasing the H3K56ac on chromatin during post-diapause development.

<p>(<b>A</b>) Morphology of embryos in control group (all 48h in 2% seawater) and test group (0h-24h in 2% seawater with NM and the following 48h in 2% seawater without NM) (<b>B</b>) Western blotting for H3K56ac in total histone extracts, chromatin, and non-chromatin fractions of control and test samples from the indicated time points. The purity of histone extracts and protein composition of chromatin and non-chromatin fractions were evaluated by Coomassie-stained gels. Tubulin was used as a loading control for non-chromatin fractions. Histone H3 was used as a loading control for chromatin fractions and total histone extracts.</p