Genome context of the EpCAM gene (indicated by a blue bar) on chromosome 2 p21.

<p>The gene is shown in blue, its CpG island in green and the amplicon in black. The amplicon sequence is given below, the ZF binding site sequence is shaded in red. This picture was generated using University of California Santa Cruz genome browser (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087703#pone.0087703-Kent1" target="_blank">[66]</a>.</p

Examples of the results of the DNA methylation analysis of the EpCAM gene promoter in SKOV3 cells.

<p>The following abbreviations were used: SKOV3 cells, untreated cells; ZF, SKOV3 cells transfected with Zinc finger construct, ZF-Dnmt3aCD, cells transfected with the Zinc finger Dnmt3a catalytic domain construct; VEC cntrl, cells transfected with empty vector; Dnmt3aCD, cells transfected with a Dnmt3aCD construct without Zinc finger; CD1, stable cell line expressing ZF-Dnmt3aCD 1; CD2, stable cell line expressing ZF-Dnmt3aCD 2. The horizontal rows indicate the CpGs in the amplicon analyzed and the vertical rows represent individual clones that were sequenced. The blue and red colors represent unmethylated CpG and methylated CpG, respectively, for white colored sites, the methylation state is unknown due to technical reasons.</p

Absence of off-target methylation in SKOV3 cells analyzed by bisulfite sequencing.

<p>Methylation of four non-target genes was analyzed (KIAA0179, DSCR3, Sumo3 and WRB). Data presentation is as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087703#pone-0087703-g003" target="_blank">Fig. 3</a>. The sequences of the regions analyzed here are given in the Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087703#pone.0087703.s001" target="_blank">Information S1</a>.</p

MOESM1 of Application of recombinant TAF3 PHD domain instead of anti-H3K4me3 antibody

Additional file 1. Supplementary figures and table

Analysis of EpCAM gene expression after targeted promoter methylation in stable cell lines.

<p>A) Example of the RT qPCR analysis of EpCAM (left) and beta actin (right) mRNA amounts in SKOV3 cells and in two independent cell lines which stably express the ZF-Dnmt3a construct (CD1 and CD2). B) Quantification of the RT qPCR analysis of EpCAM expression as shown in panel A. We carried out two independent RNA preparations each analyzed in three technical repeats. The image shows the average of both results, the error bars indicate the standard error. C) Example of the Western blot analysis of EpCAM expression in SKOV3 cells and the CD1 and CD2 stable cell lines (upper panel). Beta actin was used as loading control (lower panel). The EpCAM and beta actin bands are marked with arrows. D) Quantification of the Western Blot analysis of EpCAM expression as shown in panel C. The figure shows an average of two independent experiments, the error bars indicate the standard deviation of the data.</p

Principle of targeted DNA methylation and gene silencing using Zinc fingers (ZF) fused to the catalytic domain of the DNA methyltransferase Dnmt3a (Dnmt3aCD).

<p>The blue bar represents the ZF binding site, unfilled lollipops represent unmethylated CpGs and filled lollipops represent methylated CpGs.</p

Down regulation of EpCAM expression inhibits the proliferation of SKOV3 cells.

<p>A) Results of CCK8 cell proliferation assays conducted with the CD1 and CD2 stable cell lines and SKOV3 cells as reference. The results plotted are from four independent experiments and the error bars indicate the standard error of the mean. B) Viable cell counting performed by Trypan blue staining. The graph represents the data from two independent experiments and the error bars indicate the standard error of the mean.</p

Additional file 1: Figure S1. of Genome and catabolic subproteomes of the marine, nutritionally versatile, sulfate-reducing bacterium Desulfococcus multivorans DSM 2059

Distribution of the 1,307 detected proteins by 2D DIGE, whole cell shotgun analysis and preparation of the membrane protein-enriched fraction of D. multivorans grown with 17 different substrates. Figure S2 Phylogenetic relationship of the class II benzoyl-CoA reductase catalytic subunit BamB and other uncharacterized aldehyde: ferredoxin oxidoreductases (AFOR) of selected Deltaproteobacteria. Figure S3 Scale model and chromosomal localization of transmembrane redox complex containing genes of D. multivorans. Table S1 Listing of locus tags of genes manually assigned to metabolic pathways and energy conservation as displayed in Figs. 2 and 3. (PDF 433 kb

Transcription start and end site enrichment using the TeloPrime Full-Length cDNA amplification kit (Lexogen).

<p>(A-C) Superimposed density plots of the PacBio Iso-Seq alignment coordinates against the 5’ and 3’ ends of their targets. TeloPrime cDNA libraries (red bars), SMARTer cDNA libraries (light blue bars), overlay of the two protocols (brown bars). (A) 1–2 kb size fraction; (B) 2–3 kb size fraction; (C) 3–6 kb size fraction. 10 kb around the annotated gene start and end coordinates are shown on the x-axis. Vertical green dashed lines highlight annotated target start and end sites.</p

<i>Arabidopsis thaliana</i> PacBio Iso-Seq output summary.

<p>cDNA was synthesised with Lexogen TeloPrime Full-Length amplification kit or the Clontech SMARTer PCR cDNA Synthesis Kit and then split into three size fractions (1–2, 2–3 and 3–6 kb), respectively. From left to right: number of transcript isoform clusters (HQ reads) assembled using the PacBio Iso-Seq pipeline; number of HQ reads aligning to the <i>A</i>. <i>thaliana</i> genome with a sequence identity > = 90%; percentage of full length HQ reads (FL) defined as the cumulative number of reads equal or larger in length than their respective target.</p