Epifluorescence microscopy.

<p>Y-ART3 line schizont stained with acridine orange (a) showed a typical multiple nuclei stage but no malaria pigment inclusion could be observed in a bright field (a'). Y-control line schizont stained with acridine orange (b) showed also a typical multiple nuclei stage but there is a large amount of malaria pigment also called hemozoin (the black inclusion) observed in a bright field (b').</p

<i>In vitro</i> chemosusceptibility of lines Y-ART3, Y-CQ1 and Y-control to methylene blue (MB).

<p><i>In vitro</i> chemosusceptibility of lines Y-ART3, Y-CQ1 and Y-control to methylene blue (MB).</p

Parasitemia (%) profile of Y-control line, selected resistant lines and release lines (inoculum 1×10⁸ parasites).

<p>Drug resistance selection led to a loss of fitness and virulence as all the selected lines became not-lethal (A). Release of drug pressure partially restores fitness and virulence; lines could become lethal again (B). Parasitemia kinetic of the strains Y-ART3 (large square), Y-CQ2 (small square), Y-control (circle) and death of mice (cross) are shown. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032620#pone-0032620-g002" target="_blank">Figure 2</a> is one representative experiment from more than 5 experiments done with each of the strains tested.</p

Alignment of predicted HDP sequence from the Y-control line cDNA sequence (<i>P. yoelii</i>) with <i>P. berghei</i> and <i>P. falciparum</i> referenced sequences (genes <i>pb000650.00.0</i> and <i>pf14_0446</i> respectively).

<p>The alignment was performed with ClustalW2 and treatment of the data with BoxShade 3.21; black and grey boxes indicate respectively identical and similar amino-acids between the 3 sequences studied. The Y-control sequence shares 97% identity with the <i>P. berghei</i> sequence and 77% with the <i>P. falciparum</i> sequence (data from ClustalW2).</p

<i>Ex vivo</i> chemosusceptibility values for quinolines.

<p><i>Ex vivo</i> chemosusceptibility values for quinolines.</p

<i>Ex vivo</i> chemosusceptibility values for not-lysosomotropes.

<p><i>Ex vivo</i> chemosusceptibility values for not-lysosomotropes.</p

Hemozoin concentrations in Y-control and selected lines.

<p>Hemozoin concentrations in Y-control and selected lines.</p

mRNA expression of <i>pymdr</i> (A) <i>pyhdp</i> (B) and <i>pygcs</i> (C).

<p>The results show relative expression in arbitrary units normalized to <i>pysts</i> expression (mean ± SEM of 3 independent experiments). *Comparison of Y-ART2, Y-ART3 and Y-CQ2 <i>vs</i> Y-control: p<0.03 by a one-way analysis of variance test and then p<0.05 by a pair-wise multiple comparison test <i>vs</i> Y-control group (Dunnett's method).</p

Experimental scheme of the 5-year drug resistance selection protocol.

<p>Experimental scheme of the 5-year drug resistance selection protocol.</p

Scheme of Heme catabolim pathways in <i>Plasmodium</i>.

<p>Pathway A: GSH mediated heme catabolism. Pathway B: hemozoin biocrystallisation heme mediated catabolism. The pathway B is the standard way of heme catobolism, in majority present in sensitive and revertant lines instead of way A should be predominant in the resistance selected lines presented here. CYT : cytoplasm; FV: food vacuole; RBC: red blood cell.</p