Mean path distance (a) and mean escape latency (b) of IS and IDA groups as a function of blocks of trials in the acquisition phase of the MWM.

<p>Mean percentage of path distance in target quadrant (c) and mean percentage of time spent in the target quadrant (d) as a function of group during the MWM retention phase (Dots illustrate individual scores of all IS (black square dots) and IDA pups (black circle dots)). Mean path distance (e) and mean escape latency (f) of IS and IDA groups as a function of sessions in the inversion phase of the MWM. Error bars represent 95% confidence intervals.</p

Impact of W102A mutations and of FLAP expression on the biosynthesis of 5-LO products.

<p>HEK293 cells expressing FLAP-HA or not were transfected with expression vectors coding for 5-LO1, the 5-LO1-W102A mutant, 5-LO∆13 or the 5-LO∆13-W102A mutant as shown. Control cells (last column) were transfected with a control pcDNA3.1 vector. HEK293 cells were then stimulated with 1 μM thapsigargin and 10 μM AA. 5-LO products were measured by HPLC as described in the Methods section and represent the sum of LTB₄, its trans isomers and 5-hydroxyeicosatetraenoic acid. Data represent means ± SEM of 3 or 4 independent experiments. *Values without a common superscript are different, p<0.05.</p

Iron status for IS and IDA guinea-pig dams¹ and pups² (M).

<p>¹ IS (<i>n</i> = 10) and IDA (<i>n</i> = 8)</p><p>² IS (<i>n</i> = 26) and IDA (<i>n</i> = 14).</p><p>Iron status for IS and IDA guinea-pig dams<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133168#t001fn001" target="_blank">¹</a> and pups<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133168#t001fn002" target="_blank">²</a> (M).</p

Median (horizontal bar) and individual scores of all IS (black square dots) and IDA pups (black circle dots) for (a) the total cell crossings, (b) the percentage of cells in the enclosure visited, (c) the percentage of time in the four corners of the enclosure and (d) the percentage of time spent in the guinea pig’s most visited cell in Session 2 of the OFT (PNd40).

<p>* Significant after Bonferroni correction for non-independent tests (<i>α</i> = 0.025).</p

The Δ-13 isoform of 5-lipoxygenase inhibits LT biosynthesis in a dose-dependant manner.

<p><b>(A)</b> Immunoblots showing expression of 5-LO1 (top band) and 5-LOΔ13 (lower band) after transfections with the indicated ratios of 5-LOΔ13/5-LO1 expression vectors, as well as the presence of FLAP-HA and β-actin as loading control in HEK293 cells. <b>(B)</b> HEK293 cells expressing FLAP-HA and transfected with the indicated ratios of 5-LO1 and 5-LO∆13 expression vectors were stimulated with 1 μM thapsigargin and 10 μM AA for 30 minutes. 5-LO products were measured by HPLC as described in the Methods section. Leukotrienes (LTs) are the sum of LTB₄ and its trans isomers. 5-HETE = 5-hydroxyeicosatetraenoic acid. <b>(C)</b> HeLa cells transfected with a 1:1 ratio of vectors expressing 5-LO1 and 5-LO∆13 were stimulated under the same conditions as in (B) and LTs and 5-HETE production were measured. Immunoblots are representative of 4 independent experiments. Data represent means ± SEM of 4 independent experiments. *Different from control p<0.05, and **p<0.01.</p

Intensity profiles of of 5-LO1 and 5-LO∆13 staining in resting and stimulated cells.

<p>HEK293 cells expressing FLAP which were transfected to express either 5-LO1 or 5-LO∆13 were stimulated with 1 μM A23187 and 10 μM AA or were incubated with their diluent (resting) for 10 minutes. Cells were then fixed and permeabilized and then incubated with rabbit anti-5-LO. Slides were then incubated with an Alexa488-conjugated secondary anti-rabbit antibody and with DAPI to visualize nuclei, and slides were then mounted. Samples were analysed by confocal microscopy and the intensity of the signals of cross-sections of cells were measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132607#pone.0132607.g005" target="_blank">Fig 5</a>. <b>Top panels:</b> The intensity of each cross section was measured at the centre of the nucleus (N), at the nuclear envelope (E) identified by the edge of DAPI staining, at 1 μm and at 3 μm from the nuclear envelope (cytoplasmic side). Values are means ± SEM, n = 9. Values in the same figure without a common superscript are different, p<0.05. <b>Bottom panel:</b> Box and whisker plots showing the distance from the centre of the nucleus of the most intense anti-5-LO staining of cross sections. The box represents the two middle quartiles of data, the vertical line in the box is the median and the whiskers represent the minimum and maximum of all the data. *Median value different from others, p<0.05. Values in all panels are from intensity cross sections of 9 cells per condition from three different fields of view, each field representing a separate experiment.</p

Birth outcomes for IS and IDA guinea-pig dams¹ (M ± SEM).

<p>¹ IS (<i>n</i> = 10) and IDA (<i>n</i> = 8).</p><p>Birth outcomes for IS and IDA guinea-pig dams<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133168#t002fn001" target="_blank">¹</a> (M ± SEM).</p

Known regulatory factors are retained in the protein sequence of 5-LO∆13.

<p>chematic of the protein sequences of the 617 amino acid 5-LO∆13 isoform (top) and of the 674 amino acid 5-LO1 isoform (bottom). All known phosphorylation sites, import and export sequences are shown to be present maintained in 5-LO∆13 despite the lack of exon 13. The kinases (MAPKAPK2, PKA and ERK) responsible for phosphorylation of the different sites are indicated as is residue W102 that is responsible for the interaction of 5-LO1 with CLP, and for the CLP-induced increase in 5-LO1 activity in cell-free assays.</p

Sub-cellular localization of the W102A mutants of 5-LO1 and 5-LO∆13 in resting and stimulated cells.

<p>HEK293 cells expressing FLAP and expressing either the 5-LO1-W102A mutant or the 5-LO∆13-W102A mutant, or which were transfected with a control vector (pcDNA) were stimulated with 1 μM A23187 and 10 μM AA or were incubated with their diluent (resting) for 10 minutes. Cells were then fixed and permeabilized and then incubated with rabbit anti-5-LO. Slides were then incubated with an Alexa488-conjugated secondary anti-rabbit antibody (green) and with DAPI (blue) to visualize nuclei, and slides were then mounted. Samples were analysed by confocal microscopy and images are presented on the left panels. The intensity of the signals could be visualized by intensity profiles (right panels) of the regions indicated by the white line on the merge images. Images are representative of three independent experiments. Scale bar represents 30 μm.</p

Phosphorylation pattern of 5-LO1 and 5-LO∆13 is different in HEK293 cells.

<p>Immunoblot analysis of resting HEK293 cells expressing either 5-LO∆13 or 5-LO∆13. All cells also expressed FLAP and CLP. After separation of proteins by SDS-PAGE, membranes were subject to western blots using anti phospho-serine 523 (S523) or anti-phospho-serine 271 (S271). Expression of 5-LO isoforms was verified by blotting using anti-5-LO (5-LO). The immunoblots show the results of 3 independent experiments.</p