Induction of apoptosis in COS7 cells by polyfosine or edelfosine.

<p>(A) Cells incubated with 10 µM of polyfosine or edelfosine for indicated times were fixed and probed for activated caspase-3, a landmark of apoptosis. After 18 h many apoptotic cells had detached from support. Confocal laser immuno-fluorescent microscopy images are shown as inverted grayscale. Bars, 50 µm. (B & C) Lysates of cells incubated with 10 µM of polyfosine or edelfosine for indicated times were assayed by Western blotting for activated caspase-3 and for a-tubulin, which served as a load control. (C) Signal intensities from four Western blots were quantified. Shown is the amount of activated caspase-3 corrected for background and normalized to the a-tubulin signal. The amount of active caspase-3 in edelfosine treated cells was set to 100.</p

MS analysis of ether glycerophospholipids from COS7 cells incubated with 50 µM polyene-lyso-ePE or -ePC for 2 or 1 h, respectively.

<p>“O” indicates plasmanyl, “P” plasmenyl lipids.</p

UV light induced phototoxicity.

<p>COS7 cells were incubated with carrier (A) or 50 µM of lyso-ether PC (B), edelfosine (C) or polyfosine (D). Living cells were imaged by phase-contrast video microscopy for at least 60 min, during which a broadband UV illumination from a second light source was constantly applied. Bars, 20 µm. Mitochondria fragmented (C & D) and disintegrated (D). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031342#pone.0031342.s006" target="_blank">Movie S1</a>.</p

Metabolism of polyfosine in COS7 cells.

<p>Cells were incubated with 50 µM of polyfosine for different times. Lipids were extracted and analyzed by TLC for fluorescent metabolites, which were identified by comigrating lipid standards.</p

Imaging of polyfosine in COS7 cells.

<p>Cells incubated with 50 µM of polyfosine for 1 h (A, D & E), 2.5 h (B), or 5 h (C) were imaged by two-photon-excited lipid fluorescence (A–C) or epifluorescence (D & E) microscopy. Merged color images show polyfosine in green (D & E) and mitochondria (D) or lysosomes (E) stained by Mitotracker or Lysotracker, respectively, in red. Polyfosine stained convoluted PM ruffles (D, arrow heads). Bars, 20 µm.</p

Metabolism of polyene-lyso-etherlipids in COS7 cells.

<p>Cells were incubated with 50 µM of c16:5-lyso-ePE (A) or c16:5-lyso-ePC (B) for different pulse times. Fresh medium was applied for chase times. Lipid were extracted and analyzed by TLC for fluorescent metabolites, which were identified by comigrating lipid standards.</p

Imaging of polyene-ether phospholipids in COS7 cells.

<p>Cells were incubated with 50 µM lyso-ePE (A & B) or lyso-ePC (C & D) for 1 h (A & C) or 1 h+2 h chase (B & D). Fresh medium was applied for chase times. Cellular metabolism generated mostly ePE (A & B) or ePC (C & D) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031342#pone-0031342-g003" target="_blank">Fig. 3 A & B</a>, respectively). Living cells were imaged using two-photon-excitation microscopy. Note, that the reticular ER staining appears less defined after the chase (B & D). Bars, 20 µm.</p

Inhibition of PC synthesis.

<p>(A) COS7 cells were incubated with 10 µM polyfosine (closed circles) or edelfosine (open circles) in the presence of [3H]-myristic acid. At indicated times lipids were extracted and analyzed by TLC for radioactive metabolites. The signal intensity of labeled PC was quantified from TLC plates. Data are mean values ± range, n = 2. Error bars smaller than symbol size are omitted. (B) COS7 cells were incubated with 50 µM polyfosine or edelfosine for 1 h. Cellular lipids were extracted and analyzed by MS. The total PC and PE intensity was normalized to the sum of all major lipids (PC, PE, SM, DG, TG, ePC and ePE). Data are mean values, n = 2, with a range of less then three percent.</p

Uptake and labeling specificity of various polyene-ether lipid precursors.

<p>The total amount of fluorescent lipids in COS7 was determined by fluorescence spectroscopy and plotted over incubation time (A). Fluorescent ePC (B) or ePE (C), derived from c16:5-alkyl-1-glycerol (B & C, closed circles) or c16:5-lyso-ePC (B, open circles) or c16:5-lyso-ePE (C, open circles), respectively, was quantified from TLC plates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031342#pone-0031342-g002" target="_blank">Figs. 2</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031342#pone-0031342-g003" target="_blank">3</a>). Results from a single experiment are displayed as background-corrected percent values of total fluorescence in the TLC lane.</p

Knock-down of PHA-4, DAF-16, SKN-1, ATG-12, or SPHK-1 affect the extended longevity of <i>hyl-1;lagr-1.</i>

<p>Cumulative survival curves of N2 and <i>hyl-1;lagr-1</i> worms grown at 20°C subjected to either empty vector control bacteria (L4440) or the indicated RNAi from the early adult stage. (A) When subjected to <i>atg-12</i> RNAi, the extended lifespan of <i>hyl-1;lagr-1</i> is normalized to the extent of <i>atg-12(RNAi)</i> control animals, P = 0.3053. (B) When subjected to <i>pha-</i>4 RNAi, the extended lifespan of <i>hyl-1;lagr-1</i> is normalized to the extent of <i>pha-4(RNAi)</i> control animals, P = 0.2369. (C) When subjected to <i>daf-16</i> RNAi, the extended lifespan of <i>hyl-1;lagr-1</i> is decreased beyond the extent of <i>daf-16(RNAi)</i> control animals, P = 0.0002. (D) When subjected to <i>skn-1</i> RNAi, the extended lifespan of <i>hyl-1;lagr-1</i> is normalized to the extent of <i>skn-1(RNAi)</i> control animals, P = 0.5476. (E) When subjected to <i>daf-2</i> RNAi, <i>hyl-1;lagr-1</i> lifespan is further extended compared to both <i>hyl-1;lagr-1</i> control animals, P<0.0001, and <i>daf-2(RNAi)</i> control animals, P<0.0001. (F) When subjected to <i>eat-2</i> RNAi, <i>hyl-1;lagr-1</i> lifespan is decreased compared to <i>hyl-1;lagr-1</i> control animals, P = 0.0002, while the lifespan of <i>eat-2(RNAi)</i> animals is extended compared to wild-type control animals, P<0.0001. (G) When subjected to <i>aak-2</i> RNAi, <i>hyl-1;lagr-1</i> lifespan is decreased compared to <i>hyl-1;lagr-1</i> animals, P = 0.0009, while no lifespan effect is seen when comparing <i>aak-2(RNAi)</i> animals to wild-type control animals, P = 0.0975. (H) When subjected to <i>sphk-1</i> RNAi, the extended lifespan of <i>hyl-1;lagr-1</i> is normalized to the extent of <i>sphk-1(RNAi)</i> control animals, P = 0.8002. For additional details about these experiments, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070087#pone-0070087-t001" target="_blank">Table 1</a>.</p