Phagocytosis is reduced in <i>E. histolytica</i> cells over-expressing H644 or EhLimA.

<p>Wild-type (WT) or transgenic cells were incubated with erythrocytes (hRBC:amoeba ratio; 100∶1) for 10 minutes, lysed, and spectrophotometrically analyzed for internalized heme at 405 nm. The data represent the mean ± S.D. of 4 experiments (**<i>P</i><0.01; *<i>P</i><0.05). Amoebae over-expressing H644 or EhLimA exhibit reduced phagocytosis of hRBCs.</p

Cycloheximide is toxic to <i>E. histolytica</i> cells over-expressing H644 or EhLimA.

<p>Transgenic trophozoites were exposed to 100 nM cycloheximide for 48 hours after which viability was assessed. The data are reported as a percent of untreated control amoebae. The data represent the mean ± S.D of 3 trials. Cycloheximide was toxic to both untransfected wild-type (WT) amoebae and <i>E. histolytica</i> transgenic cells. Therefore, cells over-expressing H644 or EhLimA do not exhibit a multidrug resistance phenotype.</p

Fluid-phase endocytosis is not reduced in <i>E. histolytica</i> cells over-expressing H644 or EhLimA.

<p>Wild-type (WT) or transgenic cells were incubated with the fluid-phase marker, FITC-dextran for 30 minutes, lysed, and analyzed for internalized FITC-dextran using spectrofluorimetry. The data represent the mean ± S.D. of ≥3 experiments. Amoebae over-expressing H644 or EhLimA exhibit normal uptake of fluid-phase marker suggesting that these cell lines do not have wide-spread defects in endocytosis.</p

Tubercidin-loaded erythrocytes are toxic to <i>E. histolytica</i>.

<p>Trophozoites were exposed to tubercidin-loaded erythrocytes two or three times (# of Treatments) and viability was assessed 24 hours or 48 hours after treatment. The data are reported as a percent of the starting number of viable amoebae before treatment. The data represent the mean ± S.D of ≥3 trials (***<i>P</i><0.001). Three treatments of <i>E. histolytica</i> cells with tubercidin-charged hRBCs resulted in the death of nearly 100% of the cells 48 hours after application of selection.</p

Isolation of cDNAs from <i>E. histolytica</i> over-expressors without (Control) and with (Selected) selection with tubercidin-charged erythrocytes.

<p>Isolation of cDNAs from <i>E. histolytica</i> over-expressors without (Control) and with (Selected) selection with tubercidin-charged erythrocytes.</p

Schematic of domains found in the hypothetical protein H644 and EhLimA.

<p>A. H644 is a hypothetical protein with a postulated molecular weight of 33.8 kDa (304 amino acids). It is predicted to have a lysine-rich region (blue hexagon), a glycosylation site (gray vertical line), a N-myristoylation site (purple vertical line), 3 casein kinase II phosphorylation sites (red flags labeled C), 5 protein kinase C phosphorylation sites (red flags labeled P), 2 tyrosine kinase phosphorylation sites (red flags labeled Y), and 2 cAMP/cGMP-dependant protein kinase phosphorylation sites (red flags labeled G). B. EhLimA has a molecular weight of 15.9 kDa (145 amino acids). It has an N-terminal LIM domain (orange pentagon), a C-terminal glutamic acid-rich region (green circle), 3 protein kinase C phosphorylation sites (red flags labeled P), and a N-myristoylation site (purple vertical line).</p

Tubercidin-loaded erythrocytes are less toxic to <i>E. histolytic</i> cells over-expressing H644 or EhLimA.

<p>Transgenic trophozoites were exposed to tubercidin-loaded erythrocytes (3 treatments) and viability was assessed 48 hours after treatments. The data are reported as a percent of the starting number of viable amoebae before treatment. The data represent the mean ± S.D of 3 trials. <i>E. histolytica</i> transgenic cells exposed to tubercidin-charged hRBCs displayed increased survival as compared to untransfected wild-type (WT) cells.</p

Tubercidin-loaded erythrocytes are less toxic to an <i>E. histolytica</i> cell line with a phagocytosis defect.

<p>Trophozoites over-expressing a GFP-tagged version of a pleckstrin homology (PH) domain from mammalian Bruton’s tyrosine kinase (GFP-PH^Btk), which exhibit reduced phagocytosis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043025#pone.0043025-Byekova1" target="_blank">[22]</a> or a control cell line over-expressing GFP alone (GFP) were exposed to tubercidin-loaded erythrocytes three times and viability was assessed 48 hours after treatment. The data are reported as a percent of the starting number of viable amoebae before treatment. The data represent the mean ± S.D of 3 trials. The phagocytic mutant was insensitive to treatment with tubercidin-charged hRBCs as evidenced by >100% survival (growth) in the presence of selection. This indicates that the selection scheme may be used to enrich for phagocytosis mutants from a population of cells.</p

qPCR confirms over-expression of H644 and EhLimA.

<p>RNA from untransfected (wild-type; WT) log phase <i>E. histolytica</i> trophozoites or from trophozoites transfected with an expression vector encoding H644 or EhLimA was used for qPCR analysis of expression. The ssRNA gene was used as a loading control. H644 and EhLimA were expressed at approximately 3.1 (±0.0)- and 5.6 (±0.4)-fold, respectively, over wild-type levels.</p

Polyribosome abundance in control and stressed cells.

<p>Total RNA from control (A), serum-starved (B), or glucose-starved (C) cells were resolved by sucrose gradient (15–45%) ultracentrifugation, which separates free ribosomes and monosomes (light fractions) from polysomes (dense fractions). The gradients were fractionated and the fractions were analyzed by UV spectrometry (254 nm). Representative profiles of at least 3 separate trials are shown. Long-term serum starvation led to a decrease in large polysome abundance.</p