CID spectrum (recorded in the negative ion mode) of (A) a dedicated PS containing 30∶2 (<i>sn</i>-1) and 20∶2 (<i>sn</i>-2) fatty acyl residues.

<p>Δ 20∶2 elimination of eicosadienoic acid, Δ 20∶2-H₂O–elimination of eicosadienoic ketene and (B) a given PE species with the following fatty acyl combinations: 28∶1/20∶2; 30∶2/18∶1.</p

31P NMR spectrum and one-dimensional TLC profile of <i>A. castellanii</i> phospholipids.

<p>Left: 242.88 MHz ³¹P NMR spectrum of the organic extract of <i>A. castellanii</i>. Assignments and chemical shifts are indicated in the spectrum. The chemical structures of a phospholipid and a lysophospholipid is shown. R stands for the different head groups, while R₁ and R₂ denote the different carbon chains in the <i>sn</i>-1 and <i>sn</i>-2 position of the glycerol back bone. Chemical structures of the head groups are shown on top of the corresponding peak. Right: One-dimensional TLC profile of <i>A. castellanii</i> phospholipids in comparison with a standard PL mixture that contains the same amounts of all indicated PL. TLC plates were developed in a solvent mixture consisting of chloroform∶methanol∶acetic acid∶acetone∶water (35∶25∶4∶14∶2, v/v/v/v/v). Abbreviations used in peak/spot assignments: CL, cardiolipin; LPC, lyso-phosphatidylcholine; LPE, lyso-phosphatidylethanolamine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin. For details see text.</p

Content (given in mol %) of the fatty acyl residues of each phospholipid class of <i>A. castellanii</i>.

<p>Data were determined by GC/MS analysis of the FAs methyl esters subsequent to alkaline hydrolysis of the corresponding lipid class. Standard deviations of all measurements are estimated to be of the order of ±5%.</p><p>*The position of double bonds was already determined in a previous paper <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101243#pone.0101243-PalusinskaSzysz1" target="_blank">[11]</a>, tr – trace (<0.5%).</p

Nile Red staining of yeast lipid particles.

<p>Visualization of membranes composed of glycerophospholipids (red emission) and neutral lipids, triacyglycerols and steryl esters, in lipid particles (green emission). Cells were cultured overnight. The media were then supplemented with either 100 µM simvastatin or buffer and the cells were further grown with shaking for two hours at 30°C. To localize neutral lipids and glycerophospholipids in yeast cells, Nile Red staining was performed. Horizontal panels: upper glycrophospholipids at 543 nm excitation and 610 nm emission, middle neutral lipids at 488 nm excitation and 515/530 nm emission, lower merge of above panels. Vertical panels: B cells cultivated in buffer, S cells incubated for 2 h in buffer with simvastatin.</p

Two dimensional chromatography of glycerophospholipids.

<p>The levels of all major glycerophospholipids were diminished by treatment with simvastatin. Panels: 1, 3, 5 glycerophospholipids from cells harbouring the wild-type yeast, or the wild-type or mutated <i>hHMGR</i> gene, respectively. Panels 2, 4, 6 glycerophospholipids from simvastatin treated cells harbouring the wild-type yeast, or the wild-type or mutated <i>hHMGR</i> gene, respectively. Abbreviations: PC phosphtidylcholine, PE phosphtidylethanolamine, PS phosphatidylserine, PI phosphtidylinositol, PA phosphtidic acid, LP lysoglycerophospholipid, FA fatty acid, NL neutral lipids.</p

Decrease in sterols and squalene after simvastatin treatment.

<p>Lipids extracted from yeast cells were subjected to alkaline hydrolysis, purified and analysed by GC/MS.</p><p>Wt, wild-type yeast; H, yeast harbouring wild-type <i>hHMGR</i> gene; h, yeast harbouring the mutated <i>hHMGR</i> gene.</p

TLC analysis of complex lipids.

<p>Effect of simvastatin treatment on glycerophospholipids, apparently not connected with mevalonate pathway. Lanes 1, 3, 5 lipids from cells harbouring the wild-type yeast, or the wild-type or mutated <i>hHMGR</i> gene, respectively. Lanes 2, 4, 6 lipids from cells treated with simvastatin harbouring the wild-type yeast, or the wild-type or mutated <i>hHMGR</i> gene, respectively. Abbreviations: SQ squalene, SM simvastatin metabolites, PE phosphtidylethanolamine, GPL glycerophospholipids, SF sphingomyeline.</p

GC/MS analysis of sterols from yeast cells.

<p>Changes in the level of ergosterol and its precursors in cells treated with simvastatin depend on the mutation in the <i>hHMGR</i> gene. Panels 1, 3, 5 sterols from cells harbouring the wild-type yeast, or the wild-type or mutated <i>hHMGR</i> gene, respectively. Panels 2, 4, 6 sterols from cells treated with simvastatin harbouring the wild-type yeast, or the wild-type or mutated <i>hHMGR</i> gene, respectively. SQ squalene, Z zymosterol, E ergosterol, F fecosterol and isomers, L lanosterol and cholesta-8,14-dien 3-ol, 4.4-dimethyl (3∃, 5∀).</p