Effect of butyrate on GSTA1/A2 mRNA levels and protein expression in intestinal epithelial cells and CrD mucosal epithelial cells challenged with LPS from <i>Escherichia Coli</i>.

<p>(<b>A–B</b>) Caco-2 cells were treated for 24 hours with butyrate and then were stimulated with EC-LPS for 4 h. (<b>A</b>) Real time PCR of GSTA1/A2 mRNA. Values are means ± s.d., n = 6. Asterisks indicate that means differ from samples cultured with medium alone and from samples cultured with EC-LPS. <i>*P</i><0.05. (<b>B</b>, <i>top line</i>) Immunoblot of GST-α. β-actin was used as loading control for blot. (<b>B</b>, <i>bottom line</i>) Densitometric analysis of the band intensity. Values are means ± s.d., n = 6. (<b>C–D</b>) CrD colonic mucosa were cultured for 4 h in the presence of medium alone, medium with EC-LPS or EC-LPS with butyrate. (<b>C</b>) Real time PCR of GSTA1/A2 mRNA. Values are means ± s.d., n = 14. Asterisks indicate that means differ from samples cultured with medium alone and from samples cultured with EC-LPS. <i>*P</i><0.05. (<b>D</b>) confocal microscopy of GST-α protein (green) in CrD colonic mucosa (n = 14). Nuclei counterstained with DAPI(blue). Scale bar, 10 µm.</p

Chemotaxis index as a function of time for neutrophils A) from donor A, B) from donor B.

<p>The vertical line indicates the time when the IL-8 solution was added to the chemoattractant reservoir.</p

Cell trajectories projected on the XY plane and referred to the same origin.

<p>A: Random motion in absence of any chemical stimulus. B: In the presence of an IL 8 concentration gradient (C₀ = 50 µg/ml) a preferential direction is qualitatively evident: the cells migrate towards the negative Y direction, i.e. in the direction of the membrane, towards the chemoattractant gradient.</p

Figure 6

<p>A: Average cell velocity components and modulus as a function of time for neutrophils from donor A. C: Average cell velocity components and modulus as a function of time for neutrophils from donor B. The vertical line indicates the moment when the IL-8 solution was added to the chemoattractant reservoir.</p

Fraction of motile cells as a function of the time for neutrophils A) from donor A, B) from donor B.

<p>The vertical line indicates the moment when the IL-8 solution was added to the chemoattractant reservoir.</p

Average cell velocity module V calculated over the entire Pre IL-8 and Post IL-8 periods for each of the 63 cells from donor A.

<p>The continuous line is the average value, the error bar corresponds to the standard deviation for each population. Data are statistically significant (p<0.0001). In the inset the same data are plotted as a numerical distribution.</p

Chemoattractant diffusion.

<p>A: Evolution of the concentration profile as a function of space and time along the chemotaxis chamber according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052251#pone.0052251.e002" target="_blank">Eq. 2</a>. B: Finite element discretization of the chemotaxis chamber. Chemoattractant isoconcentration curves are superimposed to the discretization mesh. C: Chemoattractant concentration, normalized with respect to the initial concentration in the reservoir (C₀) as a function of time at a distance of 2, 3 and 5 mm from the membrane. Dashed lines are the semi-infinite slab approximation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052251#pone.0052251.e002" target="_blank">Eq. 2</a>), continuous lines are the finite element simulations, circles are the experimental measurement of FITC-dextran concentration by mean grey intensity. Inset: Mean Gray Level measurement of epifluorescence images as a function of dextran concentration.</p

Images acquired at 5 consecutive focus positions within the collagen gel have been projected on the XY plane.

<p>The circles indicate the position of cells at two different times. Only the cells enclosed in a black circle move, while the white ones do not significantly change their position over the entire experiment. A: Time = 0. B: Time = 110 minutes. The complete trajectories described by motile cells are shown. The arrow indicates the direction of the chemoattractant concentration gradient ∇C.</p

Chemotaxis chamber.

<p>A: In the exploded view rendering of the chamber all the components are individually visible. B: In the assembled rendering, the membrane, sandwiched between two aluminum frames, is housed in the chamber, separating the sample well and the chemoattractant reservoir. C: Typical collagen gel morphology in confocal microscopy (63×, oil). Image size is 50 microns.</p

DPAP3 is secreted at the time of egress.

<p>(<b>A</b>) C2-arrested schizonts (DPAP3-HA) were either left on C2, treated with E64 after C2 wash out, or allowed to egress for 1 h in the presence of FY01. Parasite pellets from free merozoites and schizonts (insoluble fraction obtained after saponin lysis), proteins precipitated from the culture supernatant, and PV and RBC cytosol components (soluble saponin fraction), were run on a SDS-PAGE. The presence of DPAP3-HA in each fraction was visualized as a fluorescent band at around 130kDa, which correspond to the band identified by WB using an anti-HA antibody (See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007031#ppat.1007031.s008" target="_blank">S4A Fig</a>). Hsp70 and BiP antibodies were used as WB markers of intracellular proteins (cytosol and ER, respectively), and SERA5 as a PV marker. (<b>B</b>) Mature DPAP3-mCh schizonts were arrested with C2 for 3 h, and egress observed by live video microscopy after C2 wash out (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007031#ppat.1007031.s016" target="_blank">S1</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007031#ppat.1007031.s018" target="_blank">S3</a> Videos). The representative still-frame pictures show DIC and mCherry signal (red) before or after PVM breakdown, and after RBCM rupture. (<b>C</b>) Quantification of mCherry signal measured on consecutive frames before and after PVM breakdown. Around 20% of the signal originates from the hemozoin autofluorescence (red line). As a bleaching control, the mCherry signal of schizonts that did not egress was quantified at the corresponding time frames. (<b>D</b>) IEM section obtained from DPAP3-GFP parasites. Close-up images of individual intracellular merozoites on the left show immunogold staining of DPAP3-GFP in close proximity to the rhoptries (green arrows) and at the apical end of merozoites (blue arrows). Images on the right show representative sections of schizonts with an intact (black arrows) or rupture PVM. Staining of extracellular DPAP3-GFP (white arrows) was only observed in schizonts lacking a PVM. Rhoptries (r), nuclei (n), and the RBCM (red arrows) are indicated. Rabbit anti-GFP and colloidal gold-conjugated anti-rabbit antibodies were used. Bar graph = 200 nm. IEM images obtained on the 3D7 control line are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007031#ppat.1007031.s007" target="_blank">S3D Fig</a>, and the uncropped IEM images for the DPAP3-GFP line in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007031#ppat.1007031.s007" target="_blank">S3E Fig</a>.</p