Data tables Statistical analyses, and figures for the different isotopologue results from <i>Legionella pneumophila</i> CsrA regulates a metabolic switch from amino acid to glycerolipid metabolism

It contains Figures S1-S7 and Tables S1-S1

Ratio between stimulatory and inhibitory motifs in <i>trypanosomatidae</i> and vertebrate genomes.

<p>The table shows the S/I ratio that corresponds to (number of stimulatory motif/number of inhibitory motif) calculated for each motif and genome. The number of motifs observed in each genome is reported as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003308#pntd-0003308-t001" target="_blank">Table 1</a>, A and B corresponding to the stimulatory motifs and C and D to the inhibitory ones. The S/I ratios are significantly higher in <i>trypanosomatidae</i> DNA than in mouse DNA, according to Wilcoxon test (p<0.5).</p><p>Ratio between stimulatory and inhibitory motifs in <i>trypanosomatidae</i> and vertebrate genomes.</p

The representation of stimulatory and inhibitory motifs in <i>L. major</i> genome is shared by other <i>Trypanosomatidae</i> genomes.

<p>The data represent the ratio of observed/expected number rO/E for each motif [stimulatory (RRCGYY and HRWCGTTN) or inhibitory (WKKVGGGG and CCNDDNNGGG)] from the analysis of <i>Trypanosomatidae</i> complete genomes (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003308#pntd-0003308-t001" target="_blank">Table 1</a>). The dotted line represent the ratio of observed/expected rO/E sequences which is 1, when no selection pressure is exerted on the genome in a neutral environment.</p

Analysis of DNA-HMGB1 complex by gel electrophoresis and Western blot.

<p><i>L. major</i> or vertebrate sonicated DNA (250 ng) were incubated with increasing amounts of HMGB1. Lane 1 shows the migration of DNA alone and lane H the HMGB1 alone. In lane 2 to 7, DNA was complexed to HMGB1, respectively at a molar ratio of 2.5, 5, 7.5, 15, 25 and 50. (<b>A</b>) Following electrophoretic migration samples were immediately blotted on a PVDF membrane and revealed with an anti-HMGB1 antibody. (<b>B</b>) Following electrophoretic migration, the gel was incubated in a solution of EtBr during 45 min to stain the DNA <b><i>(left)</i></b> then blotted and revealed with anti-HMGB1 antibody <b><i>(right)</i></b>. (<b>C</b>) Sonicated <i>Trypanosomatidae</i> DNA alone (250 ng) (lane 1) or complexed with HMGB1 at a molar ratio of 25 and 50 (lanes 6 and 7) were analysed by electrophoresis with the procedure described in <b>B</b>. Free HMGB1 (25 kDa) did not migrate in the 1% agarose gel.</p

Competition with vertebrate DNA prevents the immunostimulatory activity of <i>L. major</i> DNA.

<p>C57BL/6 BMDCs were stimulated <i>in vitro</i> for 6 h with 10 µg of <i>L. major</i> DNA (<b>A–B</b>) or <i>T. cruzi</i> DNA (<b>C</b>), alone or with 10 µg of vertebrate DNA. Cytokines production was measured by ELISA in the supernatants of cultures. (<b>A and C</b>) The data represent the mean and SEM of three independent experiments. Significant differences are indicated (*, p<0.05; **, p<0.01; ***, p<0.001). (<b>B</b>) The percentage of inhibition of BMDCs activation obtained by adding increasing amount of vertebrate DNA (mouse, pig or sheep) to <i>L. major</i> DNA. Percentage (%) of inhibition  =  [100-{cytokines production by <i>L. major</i> with vertebrate DNA/cytokines production by <i>L. major</i> DNA alone}]×100. The results represent the mean and SEM of three independent experiments.</p

Stimulation of human plasmacytoid dendritic cells by <i>L. major</i> and vertebrate DNA.

<p>Human plasmacytoid cell line (Gen2.2) was stimulated by <i>L. major</i>, mouse and human DNA alone <b><i>(left)</i></b> or complexed with DOTAP <b><i>(right)</i></b>. In the indicated lanes, cells were treated with chloroquine (20 µM) before <i>L. major</i> stimulation. Expression of the indicated cytokines was determined by real time RT-PCR. The mRNA expression levels were normalized to the expression of the HPRT gene and calculated as the n-fold difference with the expression in unstimulated cells. The results represent the mean and SEM of three independent experiments (*p<0,05, **p<0,01).</p

Retinal glia rapidly phosphorylate ERK1/2 after RGC axotomy.

<p>(<b>a–d</b>) p-ERK immunoreactivity in neurons (<b>a</b>) In uninjured retinas there is expression of ERK1/2 in all cellular compartments, both in neuronal and in non-neuronal cells, detected with an antibody to total ERK1/2. Total ERK1/2 staining continued to be expressed to the same level in retinas at days 1 and 3 post-axotomy (data not shown). (<b>c</b>) In uninjured retinas ERK1/2 is phosphorylated in RGCs. (<b>b</b>) At day 1 after ON axotomy, there was a robust increase of p-ERK1/2 immunoreactivity in putative glial cells in the GCL, INL, and both plexiform layers. Note the relative reduction of p-ERK1/2 in the GCL versus control normal retinas. (<b>d</b>) At day 3 after ON axotomy both the soma and the processes of retinal glia are robustly labeled with p-ERK1/2. Note that, unlike what was shown for p-Akt, the glial fibers with p-ERK1/2 point both towards the injured RGCs and towards the photoreceptors (full arrows). Arrowheads point to the soma of glia. (<b>e,f</b>) p-ERK immunoreactivity in glia. (<b>e</b>) p-ERK is strongly expressed in the end-feet, somas and processes of Müller cells expanding throughout the PhR layer rapidly after axotomy, as shown by co-localization of p-ERK with CRALBP. (<b>f</b>) The expression of p-ERK was low in astrocytes, as revealed by the weak co-localization of p-ERK with GFAP. GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; PhR: photoreceptor layer. Scale bar, 30 µm.</p

Selection for inhibitory and against stimulatory TLR9 motifs in vertebrate genome.

<p>(<b>A</b>) Poly G₈ (GGGG)₂, stimulatory GACGTT and inhibitory TTAGGG motifs were quantified in <i>L. major</i> and mouse genomes. Stimulatory GTCGTT motif was quantified in <i>L. major</i> and human genomes. The datas are represented as copy number per 10⁶ bases pair ( = copy number/genome size (bp)*10⁶). (<b>B</b>) Stimulatory (RRCGYY and HRWCGTTN) and inhibitory (WKKVGGGG and CCNDDNNGGG) motifs were quantified in each <i>L. major</i>, mouse or human chromosome. They are represented as the ratio of observed/expected sequences rO/E, as indicated in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003308#pntd-0003308-t001" target="_blank">Table 1</a>. For each chromosome, the ratio is represented by a single symbol. Significant differences between <i>L. major</i> and vertebrate chromosomes are indicated (***, p<0.001). The dotted line represent the ratio of observed/expected rO/E sequences which is 1, when no selection pressure is exerted on the genome in a neutral environment.</p

Treatment with PD98059 after axotomy inhibits p-ERK in glial cells and delays RGC death.

<p>p-ERK immunoreactivity in axotomized retinas from eyes treated with vehicle or PD98059 (<b>a</b>) retinas collected 3 days post-axotomy and (<b>b</b>) retinas collected 5 days post-axotomy. RGCs were labeled with FG prior to axotomy. In vehicle-injected eyes, p-ERK1/2 was increased in glial cells. In PD98059-injected eyes, p-ERK staining was reduced in the somata and in processes of Müller cell throughout all retinal layers. Scale bar, 30 µm. GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; PhR: photoreceptor layer. (<b>c</b>) Quantification of p-ERK intensity in vehicle or PD98059 treated eyes 3 days and 5 days post-axotomy (n = 4), * p<0.01 and ** p<0.001. (<b>d</b>) Representative pictures of FG-labeled RGCs in axotomized retinas treated with PD98059 or vehicle. Retinas were flat-mounted at day 7 post-axotomy and RGCs were quantified as described. The histogram is the quantification of FG-labeled RGCs in uninjured retinas and in axotomized retinas treated with PD98059 or vehicle, 7 days post-axotomy. Total of 48 images taken from n = 4 flat-mounted retinas per group. Values are number of RGC per mm², * p<0.01.</p

After axotomy some RGCs increase their DNA content.

<p>(<b>a</b>) RGCs were labeled with FG, and the retinas were flat-mounted on cover-slips and the DNA was stained with PI. Uninjured control and day 5 post-axotomy retinas were analyzed by SBC to quantify DNA intensity in FG-labeled RGCs. Axotomy significantly increases the percent of hyperploid RGCs, *** p<0.01. (<b>b</b>) Representative photomicrographs of flat-mounted retinas with images of FG and PI. The surrounding PI-labeled cells that are FG-negative, likely glia, are not RGCs by morphology and the fact that they are not labeled with FG. This PI-labeled population serves as background control. In each picture, the big white rectangle is an enlarged photomicrograph of the small white rectangle. Scale bar, 60 µm. FG: fluorogold; PI: propidium iodide.</p