NADPH-dependent 3-hydroxyacyl-CoA dehydrogenase activity in WT and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cells.

1<p>WCE, whole cell exctract.</p>2<p>glyco, partially purified glycosome fraction.</p>3<p>Mean ± SEM of n experiments (mU/mg of protein).</p>4<p>+gluc: cells cultured in SDM79 containing 10 mM glucose.</p>5<p>−gluc: cells cultured in glucose-depleted SDM79GluFree.</p><p>NADPH-dependent 3-hydroxyacyl-CoA dehydrogenase activity in WT and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cells.</p

Phenotypic analysis of Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cell.

<p>(A) growth curve of WT and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cell knock cells in glucose-rich (SDM79 with 10 mM glucose) or glucose-free (SDM79GluFree) conditions. (B) Global protein abundance in the partially purified glycosome fraction of WT (x-axis) and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cell knock cells (y-axis). Each protein identification is presented by a point at log₁₀ of normalized peptide count values taken from the proteome data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114628#pone.0114628.s004" target="_blank">S4 Figure</a>. Proteins on the dashed grey line have identical normalized peptide counts in both samples; the grey lines represent a 2-fold abundance in one condition.</p

TAG species analysis and uptake of labeled oleate.

<p>(A) Dominant TAG species in procyclic <i>T. brucei</i> cells identified by ESI/MS/MS after oleate feeding for three days (black columns) or in the control (white columns). For a complete list of TAG species detected see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114628#pone.0114628.s001" target="_blank">S1 Figure</a>. The nomenclature 54:X indicates the total carbon number of all three acyl chains and the sum of all unsaturated double bonds within the acyl chains. (B) Uptake kinetics upon growth in the presence of radiolabeled oleate for up to 8 h. The incorporation of ¹⁴C oleate into lipid species was quantified by HPTLC and a Storm 860 phosphorimager. PPL, phospholipids; TAG, triacylglycerol; SE, Steryl-esters; DAG, diacylglycerol.</p

LD and TAG turnover in WT and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cells.

<p>Cells were fed with oleate in glucose-rich SDM79 medium for three days, and after oleate withdrawal samples were taken at the time points indicated. (A) WT cells stained with BODIPY and analyzed by flow cytometry (left y-axis). Error bars represent the SEM of independent replicates (n = 3). The growth curve is given as dashed line (right y-axis). (B) Growth curve and sampling time points (arrows) for the experiments in panels (C) and (D). Total TAG content was determined in triplicate by HPTLC and densitometry in WT (C) and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> (D) cells. Error bars represent the SEM of independent replicates (n = 3). The calculated values (filled symbols) account for dilution of LDs or TAG content by cell division, based on the matched growth data.</p

Quantification of the oleate-induced lipid droplet formation.

<p>(A) BODIPY 493/503 stained LDs were counted in stacks of confocal laser scanning microscopy (CLSM) images; the average number of LDs per cell is given after oleate feeding (black column) or in the control (white column). (B) Distribution of LD numbers per cells in the population after oleate feeding (black columns) or in the control (white columns). (C) Quantification of BODIPY-stained LDs by flow cytometry after oleate feeding (black column) or in the control (white column). BODIPY 493/503 preferentially stains nonpolar lipids. Error bars give the SEM (n = 3) of values normalized to the control. (D) Quantification of TAG content by HPTLC and densitometry after oleate feeding (black columns) or in the control (white columns). Values are normalized to the control.</p

Oleate feeding stimulates lipid droplet formation in procyclic <i>T. brucei</i> cells.

<p>Staining of lipid droplets with nile red (A) or BODIPY 493/503 (B) was as detailed in experimental procedures. Myriocin treatment (0.5 µM for 24 h) was included for comparison to a previous report <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114628#pone.0114628-Bird1" target="_blank">[36]</a>. An example of several experiments is shown.</p