the Molecular Signatures Database (MSigDB), on which GSEA is based, contains mostly human and rodent gene sets, we used the human ortholog gene names/annotations for this analysis. Human orthologs of the whole microarray probe set were assigned using Biomart [http://www.ensembl.org/biomart/index. html]. GSEA was performed using the complete dataset whereas 5961 genes, i.e. those for which a human ortholog was identified, could be associated with gene sets from the database. The gene sets of the MSigDB (v3.0) databases C2 (Gene sets collected from various sources such as online pathway databases, publications in PubMed, and knowledge of domain experts) and C5 (Gene sets of Gene Ontology terms) were used for the analysis. AChE activity assay Zebrafish embryos were exposed with different APM concen- trations for 24 h (26–50 hpf). 30 embryos were used for AChE activity assay and were performed as previously described [53]. Non-linear modeling was performed from the plot of AChE activities with JMP 8.0 software (SAS, Cary, NC). Quantitative real-time PCR analysis Total RNA was extracted from 50 control, mutant or exposed zebrafish embryos at 50 hpf. 2 mg of total RNA was reversely transcribed with RevertAidTM H Minus Reverse Transcriptase (Fermentas) according to the manufacturer instructions. qPCR was carried out using Step-One-Plus PCR System (Applied Biosystems) and we used the SensiMixTM SYBR with ROX as passive reference (Bioline). qPCR experiments were performed with three independent biological replicates and each single run was performed in three replicate samples. Statistical analysis of qPCR experiments were performed with one-way ANOVA followed by Dunnett’s post test using GraphPad Prism 5.0 software (GraphPad Software, San Diego California USA). Primer sequences are given in Table S1. Relative expression levels were determined by using the DDCt method [54]. Cloning, in situ hybridization, immunohistochemistry, and microscopy The open reading frame (ORF) of hspb11 was amplified with ORF primers (Table S2), cloned into pCRII-blunt vector (Invitrogen) and was verified by sequencing. This construct was used for the synthesis of in situ hybridization probes. GFP constructs were amplified with F1-MOATG-gfp/R1-GFP primers for atg-gfp and F2-MOUTR-gfp/R1-GFP primers for utr-gfp (Table S2) using pEGFP-N1 (Clonetech) vector as template. Construct for the rescue experiment was generated by using mmhspb11-F/hspb11-ORF_Rev primers (Table S2) and hspb11-ORF-pCRII vector as template. Products were cloned into pCRII blunt vector (Invitrogen) and subcloned into pCS2P+. We performed whole-mount in situ hybridization (WISH) and immunohistochemistry as described previously [55,56]. We used the monoclonal antibody directed against slow muscle myosin (F59). The F59 monoclonal antibody developed by Frank E. Stockdale was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. WISH was performed with ,50 embryos per treatment if not stated elsewhere. Birefringence was analyzed with a stereomicroscope (MZ16F; Leica, Wetzlar, Germany) as described previously [5]. A Leica compound microscope (DM 5000B) and LCS software (Leica, Wetzlar, Germany) were used to analyze antibody staining. Muscle Activity Dependent Expression of hspb11 Morpholino knockdown and mRNA injection Morpholinos (Gene Tools, LLC, Philomath, OR) were dissolved in water and injected at the following concentrations: 0,25 mM – 1 mM MO(ATG)-hspb11 (59- TCGGGCAAAG- CATCTTCAGTGGATT- 39); 0,25 mM–1 mM mmMO-hspb11 (59-TCcGGgAAAGgATCTTCAcTGcATT-39) was a 5 base mismatch control for MO(ATG)-hspb11; 0,25 mM–0,5 mM MO(59UTR)-hspb11 (59- TTTGCTGTTGAGCTGTTTGGC- TTCT- 39); 0,25 mM–1 mM MO-p53 (59-GACCTCCTCTC- CACTAAACTACGAT-39). 59 capped mRNAs were transcribed from linearized vectors using the mMESSAGE mMACHINE SP6 Kit (Applied Biosystems/Ambion, Austin, TX). Injection needles were pulled from borosilicate glass capillary tubes with filament (GC100F-10; Warner Instruments, LLC, Hamden, CT) using a Narishige micropipette puller (Tokyo, Japan). Embryos were injected with the Eppendorf FemtoJet (Hamburg, Germany) through the chorion into the yolk compartment at the one-cell stage. Supporting Information Figure S1 Acute toxicity of AMP, DMB and DNP in zebrafish embryos. Dose response curves were determined by recording mortality in 2–50 hpf exposed embryos. Non linear regression modeling was performed with SigmaPlot version 11 (Systat Sofware Inc., San Jose, California) using the Hill 4 parameter equation (f = y0+a*x‘b/(c‘b+x‘b)). (TIF) Figure S2 AChE enzymatic activity in response to various APM concentrations. AChE enzyme activities are expressed as fold change of controls (based on specific activity). (n = 3, exposures from 26–50 hpf). (TIF) Figure S3 Heat map of a hierarchical cluster analysis of genes significantly differentially expressed in zebrafish embryos. Embryos were exposed for 24 h (26–50 hpf) or 48 h (2–50 hpf) to azinphos-methyl (APM), 1,4-dimethoxybenzene (DMB) or 1,2-dinitrophenol (DNP). Genes with significantly altered expression were identified using SAM (TM4 software suite). Treatments were performed by two separate series of experiments, one for the 24 h exposure and one for the 48 h exposure. C24 and C48 are control samples that refer to these separate experiments. Data represent the log2-ratio of each treatment or control to the average of control levels (either C24 or C48). Numbers 1–4 indicate different independent biological replicates. The cluster of genes, which are specifically regulated by APM, is marked by orange lines. (TIF) Figure S4 qPCR analysis of hspb11, pdlim3b and socs3a expression in zebrafish embryos. Embryos were exposed from 26–50 hpf to different AChE inhibitors. (A) Propoxur, (B) disulfoton, and (C) galantamine. (D) 4-nitrophenol served as unspecific (non-acetylcholinesterase inhibiting) control. Concen- trations are given in mM if not differently labeled. Bars represent the relative gene expression as fold change of the respective untreated control as mean 6 standard deviation of three replicate exposures. Control = ctrl. * P,0.05. (TIF) Figure S5 Muscle pioneer specific expression analysis of hspb11 and eng2a expression. (A) Transcripts of hspb11 mRNA are localized in muscle pioneers of wildtype or smu heterozygous embryos (+/?). (B) smu2/2 embryos lack muscle PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e29063