Plasma fatty acid distribution (%) at day 10 post infection.

<p>^∞ PI: Peroxidability index of lipids. The value is calculated based on the relative oxidation rate of unsatured fatty acids as follows:</p><p>PI = (%monoenoic x 0.025)+(%dienoic x 1)+(%trienoic x 3)+(%tetraenoic x 4)+ +(%pentaenoic x 6)+(hexaenoic x 8)</p><p>*p<0.05;</p><p>**p<0.01;</p><p>*** p<0.0001 vs CTR</p><p>n = 5</p><p>Plasma fatty acid distribution (%) at day 10 post infection.</p

Alteration in the lipid profiles of the lungs from uninfected, <i>Pb</i>NK65 or <i>Pc</i>AS infected mice.

<p>Mice infected with <i>Pb</i>NK65 (treated or not intraperitoneally with DEX 80mg/kg) or <i>Pc</i>AS were perfused and dissected at day 8 and 10 post infection. The total content of PL (panel A), ChoE (panel B), PC (panel C) /mg protein were determined as described in Materials and Methods. n = 6 mice for each condition; *p<0.05; **p<0.01; ***p<0.0001 vs CTR; °p< 0.05; °°p<0.01; °°°p<0.0001 vs <i>Pc</i>AS for each time point; $ p<0.05 <i>Pb</i>NK65-DEX vs <i>Pb</i>NK65 day 10. PL = phospholipids; ChoE = Cholesterol Esters; PC = phosphatidylcholine.</p

MOESM1 of Differential induction of malaria liver pathology in mice infected with Plasmodium chabaudi AS or Plasmodium berghei NK65

Additional file 1. ALT and AST determination in mice infected with P. berghei NK65 or P. chabaudi AS. C57BL/6J mice were injected intraperitoneally with 104 erythrocytes infected with P. berghei NK65 or P. chabaudi AS. Serum levels of AST (panel A), ALT (panel B) and the AST/ALT ratio (panel C) were determined at day 8 and 10 post infection according to manufacturer’s protocol (Teco Diagnostics, California, USA). n = 3-6 mice for each time point and strain, additional data can be found in [16]. *p < 0.05; **p < 0.01 versus control

Disease course in mice infected with <i>Pb</i>NK65 or <i>Pc</i>AS.

<p>C57BL/6J mice were injected intraperitoneally with 10⁴ erythrocytes infected with <i>Plasmodium berghei (Pb</i>NK65) or <i>P</i>. <i>chabaudi</i> AS (<i>Pc</i>AS). Peripheral parasitemia (panel A) and weights of the left lungs (panel B) were determined at day 6, 8 and 10 post infection (n = 6–8 mice for each time point and strain). (panel C) Representative pictures of right lungs (not perfused) from uninfected (CTR) or infected mice (day 10 post infection). (panel D) Hz content in lung tissue (pmole Hz /mg lung tissue) at day 10 post infection. *p<0.05; **p<0.01; *** p<0.0001 vs CTR; °°°p< 0.01 <i>Pb</i>NK65 vs <i>Pc</i>AS.(n = 6–8 mice for each time point and strain).</p

Alteration in the lipid profile of plasma from uninfected, <i>Pb</i>NK65 or <i>Pc</i>AS infected mice.

<p>PL (panel A) and neutral lipid content (panel B) of plasma of infected and uninfected mice at day 10 post infection were determinated as described in Materials and Methods.Cho = Cholesterol, ChoE = Cholesterol esters, TG = Triacylglycerols. *p<0.05; **p<0.01 vs CTR; °p<0.01 <i>Pb</i>NK65 vs <i>Pc</i>AS. n = 8–12.</p

Alterations in the lipid profile of the pulmonary surfactant of <i>Pb</i>NK65 or <i>Pc</i>AS infected mice.

<p>(Panel A) Large aggregate (LA) and (Panel B) small aggregate (SA) fractions of pulmonary surfactant were isolated from the BAL fluid of control mice and mice infected with <i>Pb</i>NK65 or <i>Pc</i>AS and the percentage distribution of PL was determined. SM = Sphingomyelin, PE = phosphatidylethanolamine, PG = phosphatidylglycerol and LPC = lysophosphatidylcholine. n = 5–7; * p< 0.05 vs CTR; ** p<0.001 vs CTR; ° p< 0.05, °° p< 0.01 vs <i>Pc</i>AS for each time point.</p

Protein content of the Large Aggregate (LA) fraction of BAL fluid from uninfected, <i>Pb</i>NK65 or <i>Pc</i>AS infected mice.

<p>Bronchoalveolar lavage (BAL) fluid samples were collected from control mice and mice infected with <i>Pb</i>NK65 (n = 10 for day 8 post infection, n = 13 for day 10 post infection), or <i>Pc</i>AS (n = 8 for day 8 or 10 post infection). The large aggregate fraction (LA) was isolated and the content of proteins was determined. *p<0.05; **p<0.01; ***p<0.001 vs CTR; °°°p<0.001 vs <i>Pc</i>AS for each time point.</p

<i>Pf</i>PKA-mediated phosphorylation contributes to immature GIE stiffness.

<p><b>A.</b> Retention in microsphilters of stage III GIEs from the B10 clone pre-incubated 30 min to 1 h at 37°C with 10 μM H89, 10 μM KT5720, 10 μM PKI-m, 10 μM compound 2, 10 μM GGTI-298 or 0,1% DMSO (Control). Error bars denote the standard error of the mean. *** and ** Highly significant differences in retention rates (*** <i>P</i> < 0.001; **<i>P <</i> 0.01); ns: non-significant differences in retention rates compared to control; n: number of experiments. Outliers are shown as open circles. <b>B.</b> Retention in microsphilters of stages III GIEs from the B10 clone and the pHL-<i>pfpka-r</i> clone cultivated with and without pyrimethamine for 15 generations. The pHL-<i>pfpka-r</i> clone was pre-incubated 15 min at 37°C with 100μM 8Br-cAMP. Error bars denote the standard error of the mean. ***Highly significant differences in retention rates (<i>P <</i> 0.001); ns: non-significant differences in retention rates; n: number of experiments. Outliers are shown as open circles. <b>C</b>. Western-blot analysis of <i>Pf</i>PKA-R expression in stage III GIE from the B10 clone and the pHL-<i>pfpka-r</i> clone cultivated in presence (+ pyr) or absence (- pyr) of pyrimethamine. Immunoblots were probed with rabbit polyclonal antibodies directed against <i>Pf</i>PKA-R and with a mAb directed against <i>Pf</i>HSP70 to normalize expression. Black star indicates the expected size for <i>Pf</i>PKA-R (50.8 kDa); Red star indicates <i>Pf</i>PKA-R with post-translational modifications. The experiment has been performed seven times. Error bars denote the standard error of the mean. *Significant differences in phosphorylation signal (*<i>P</i> < 0.05); ns: non-significant differences in phosphorylation signal. <b>D</b>. Quantitation of signal intensities in panel C using Quantity One software (BioRad). Analysis shows a 1.6-fold increase in <i>Pf</i>PKA-R expression in the pHL-<i>pfpka-r</i> clone (+ pyr) compared to B10. Decrease of <i>Pf</i>PKA-R expression in absence of pyrimethamine (- pyr) indicates a loss of episomal expression of the <i>Pf</i>PKA-R protein. <b>E.</b> Immunofluorescence analysis of stage III GIE from the B10 clone and the pHL-<i>pfpka-r</i> clone cultured for 15 generations in the presence (+ pyr) or absence (- pyr) of pyrimethamine. Infected erythrocytes were stained with anti-<i>Pf</i>PKA-R antibodies followed by anti-rabbit Alexa 594-conjugated IgG. Pictures were taken under identical exposure conditions. The bars represent 2 μm.</p

GIE filterability is dependent on cAMP concentration.

<p><b>A.</b> cAMP concentration drops in mature GIE. The total intracellular cAMP concentration was measured in stage III and V GIE using a competitive immunoassay for the quantitative determination of cAMP. GIE were harvested by magnetic isolation and aliquots of 6.10⁶ cells were assayed in duplicate wells. The assay was carried out three times. Error bars denote the standard error of the mean. **Highly significant difference compared to stage III GIE (<i>P <</i> 0.01). <b>B</b>. Western-blot analysis of PKA-R expression in MACS-purified stage III and stage V GIE (5.10e6 parasites/lane). Immunoblots were probed with rabbit polyclonal antibodies directed against <i>Pf</i>PKA-R and with a mAb directed against <i>Pf</i>HSP70 to normalize expression. Quantity One (BioRad) analysis shows that <i>Pf</i>PKA-R levels were not significantly different between stage III and stage V. <b>C</b>. Retention rates in microsphilters of stage III GIE (dark grey), stage V GIE (light grey) and uninfected red blood cells (uRBC, black) pre-incubated 15 min at 37°C with different concentrations of 8Br-cAMP. Error bars denote the standard error of the mean. Outliers are shown as open circles. **Highly significant differences in retention rates compared to control without 8Br-cAMP (<i>P <</i> 0.01); ns: non-significant differences in retention rates compared to control; n: number of experiments.</p

Sildenafil impairs mature GIE filterability.

<p><b>A</b>. Stage V GIE were harvested by magnetic isolation and incubated at 37°C for 30 min with 100 μM sildenafil, or 0.1% DMSO (Control). The total intracellular cAMP concentration was measured on aliquots of 6.10⁶ cells in duplicate wells. The assay was carried out five times. Error bars denote the standard error of the mean. **Highly significant differences in retention rates compared to control (DMSO) (<i>P <</i> 0.01). <b>B.</b> Graphical representation for the proportion of GIE showing a regular (light green), or deformed (dark green) shape in a population of paraformaldehyde-fixed GIE, as they flow through the microsphilters after pre-incubation at 37°C for 30 min with 100μM sildenafil, or 0.1% DMSO (Control). <b>C</b>. Differential interference contrast images of paraformaldehyde-fixed GIE, as they flow through the microsphilters. <b>D</b>. Retention rates in microsphilters of stage V GIE (light grey) and uninfected red blood cells (uRBC, dark grey) pre-incubated 30 min at 37°C with different concentrations of sildenafil. Error bars denote the standard error of the mean. Outliers are shown as open circles. *** and **Highly significant differences in retention rates compared to control (**<i>P <</i> 0.01; ***<i>P <</i> 0.001); ns: non-significant differences in retention rates compared to control. n: number of experiments.</p