Gene targeting by <i>Ppapt-KO</i> mismatch vectors in <i>Ppsrs2-KO</i>.

<p>Gene targeting by <i>Ppapt-KO</i> mismatch vectors in <i>Ppsrs2-KO</i>.</p

Up-regulated genes annotated as DNA helicases.

<p>Up-regulated genes annotated as DNA helicases.</p

Time-course of the transcriptional response of selected DNA repair genes.

<p>Tissue was incubated in BCDAT medium supplemented with 200ng.ml^-1 for 1, 4, 8, 16 and 24 hours prior to harvest. RNA was extracted for determination of transcript levels by real-time qPCR. The fold changes in transcript abundance relative to control (drug-free) treatments are shown (means ± SEM). <b>(A)</b> Rad51-1 mRNA (black bars); Rad51-2 mRNA (grey bars). <b>(B)</b> Ku70 (black bars); Ku80 (grey bars). <b>(C)</b> PARP-1 (Pp3c22_13240V3.1) (black bars); PARP-2 (Pp3c8_17220V3.1) (grey bars); <b>(D)</b> SRS2-like helicase (Pp3c1_29170V3.1) (black bars); Alc1-like helicase (Pp3c10_6710V3.1) (grey bars).</p

Gene targeting efficiency in mutant lines.

<p>Gene targeting efficiency in mutant lines.</p

Differentially regulated genes annotated as cell-cycle associatedFold change.

<p>Differentially regulated genes annotated as cell-cycle associatedFold change.</p

Up-regulated DNA repair and replication genes.

<p>Up-regulated DNA repair and replication genes.</p

Growth responses of <i>P</i>. <i>patens</i> to treatment with bleomycin.

<p><b>(A) Chronic exposure.</b> Tissue explants were inoculated on BCDAT medium supplemented with bleomycin at 0, 0.32, 1.6, 8, 40, 200 and 1000 ng.ml^-1 bleomycin as indicated, and incubated for 10 days under standard growth conditions. <b>(B) Chronic exposure.</b> Colony areas (mean ± SD) for plants exposed to bleomycin. Colony areas in the plates illustrated in Fig 1a were determined by image analysis of digital photographs of the individual plates, and are presented as the mean colony areas normalized to the area of the culture dish for each treatment. <b>(C) Acute treatment.</b> Tissue was incubated for 24h in BCDAT liquid medium supplemented with bleomycin at the concentrations indicated. Explants were then inoculated on drug-free medium and incubated under standard growth conditions for 23d and photographed at 9, 16 and 23d for image analysis. Mean colony areas (± SD, n = 12) were normalized to the area of the culture dish.</p

<i>ELMO1</i> methylation and expression in RA FLS and synovium.

<p>(<b>A</b>) A summary of DNA methylation at the <i>ELMO1</i> promoter regions from our previous study of FLS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124254#pone.0124254.ref004" target="_blank">4</a>] and the location of the <i>ELMO1</i> RA GWAS SNP. The methylation levels of each of the CGs on the bead array that are within the <i>ELMO1</i> promoter region (-2500bps to +500bps from TSS) are shown. Error bars represent the standard error of the mean (SEM) and stars highlight significantly differentially methylated CGs. The locations of the TSSs are indicated with arrows and the transcript variant numbers of the RefSeq genes that are transcribed from that TSS are shown. Comparisons are shown between RA, osteoarthritis (OA) and normal (NL) FLS. (<b>B</b>) Western blots showing the expression of and GAPDH in RA and OA synovial tissue. First lane shows positive control, and subsequent lanes show <i>ELMO1</i> protein levels in whole RA and osteoarthritis (OA) synovium (n = 4 each). There was no significant difference in overall <i>ELMO1</i> expression. (<b>C</b>) Immunohistochemistry for <i>ELMO1</i> expression in RA and OA synovial tissue. Note prominent staining in synovial intimal lining and sublining perivascular regions (brown color). Osteoarthritis synovium had a similar distribution. Left panels shows anti-<i>ELMO1</i> antibody. Right panels shows control IgG. Tissues were lightly counterstained with hematoxlin. Original image at 200x magnification.</p

<i>ELMO1</i> promotes cell migration and invasion by RA FLS.

<p>(<b>A</b>) Cell migration in the wound closure assay shows that <i>ELMO1</i> knockdown with siRNA decreases cell migration whereas the control or ELMO2 siRNAs do not. Mean and SEM was calculated from 5 experiments for <i>ELMO1</i> and 3 experiments for ELMO2. (<b>B</b>) Cell invasion assay shows that <i>ELMO1</i> promotes the movement of FLS into extracellular matrix. Levels were calculated in the presence and absence (MED) of PDGF and with control and <i>ELMO1</i> siRNA. Mean cell number and SEM was calculated from 10x field-of view images and then normalized to control. (<b>C</b>) Fields of view showing FLS invading through a Martigel layer. Note the decreased number of invading cells when the FLS are pre-treated with <i>ELMO1</i> siRNA.</p

<i>ELMO1</i> regulates RAC1 in RA FLS.

<p>(<b>A</b>) Western blots showing RAC1 and Rac1-GTP levels in RA FLS following PDGF stimulation with <i>ELMO1</i> and control siRNA. RAC1 activation was determined as described in Material and Methods. Cells were assayed from 0 to 15 min after they were stimulated with PDGF. (<b>B</b>) Quantification of the RAC1 assay. Mean and SEM was calculated from 3 experiments. The ratio of Rac1 and Rac1-GTP in RA FLS following PDGF stimulation with <i>ELMO1</i> and control siRNA is shown and demonstrates decreased RAC1 activation when FLS are <i>ELMO1</i>-deficient.</p