The Origin of NO in P. tricornutum and Its Interplay with Calcium

<div><p>(A) Intracellular localization of DAF-FM-derived fluorescence (green) compared with DAPI-staining (blue) and chlorophyll (Chl) autofluorescence (red) in P. tricornutum.</p> <p>(B) NOS enzymatic activity in cell-free extracts induced by DD (66 μM [10 μg/ml]), in the presence or absence of calcium.</p> <p>(C) Ca²⁺ transients in response to addition of 1, 3, and 5 μg/ml (6.6, 19.8, and 33 μM) DD, depicted in blue, pink, and green respectively and of 10 μg/ml (65 μM) (<i>2E</i>)-decenal (yellow) in transgenic P. tricornutum cells expressing the calcium-sensitive photoprotein Aequorin. Addition is indicated by arrow. Data in (B) are means plus standard deviation from four experiments. Representative data from at least four experiments are shown in (A) and (C). Scale bar represents 5 μm.</p></div

Sublethal DD Concentrations Can Induce Resistance to Lethal Doses in P. tricornutum

<div><p>Cells were pretreated with 0.1 μg/ml (660 nM) DD for 2 h prior to subsequent addition of 2 μg/ml (13.2 μM) DD (blue) and compared to a single dose treatment of 2 μg/ml (13.2 μM) DD (green).</p> <p>(A and B) Cell death was assayed by flow cytometry both qualitatively in cytograms (A) and quantitatively (B) using Sytox Green at the indicated time points.</p> <p>(C) Cell growth curves following resuspension of pretreated and non-pretreated cells in DD-free fresh medium 60 h after the 2 μg/ml (13.2 μM) DD treatment. The time scale indicates the days following resuspension. Inset shows photograph of the two cultures taken 2 wk after resuspension starting from an initial inoculum of 5 × 10⁴ cells/ml.</p> <p>Representative data from at least five experiments are shown in (A–C).</p> <p>a.u., arbitrary units.</p></div

DD-Dependent NO Production Induces Cell Death

<div><p>(A) Micrographs of P. tricornutum cells treated with DD (66 μM [10 μg/ml]) for 4 h, which resulted in 90% cell death (assayed by Sytox Green fluorescence). Chlorophyll autofluorescence (shown in red) was significantly reduced in Sytox-positive cells, giving a further indication of cell death.</p> <p>(B–D) Quantification of cell death kinetics induced by DD or (<i>2E</i>)-decenal (B), SNP (C), and NMMA added prior to DD application (D). Data in (B–D) are means plus standard deviation from four experiments. Representative data from four experiments are shown in (A). Experiments shown in (B–D) were performed by flow cytometry. Abbreviations are as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040060#pbio-0040060-g001" target="_blank">Figure 1</a>. Scale bar represents 5 μm.</p></div

The Role of NO and Calcium in Acclimation to Sublethal Doses of DD

<div><p>(A) Cytosolic calcium transients in transgenic P. tricornutum cells expressing Aequorin. Left panel shows calcium response in non-pretreated cells (green) following addition of 13.2 μM (2 μg/ml) DD (indicated by arrow), whereas right panel shows the calcium signature of cells that had been pretreated with 660 nM (0.1 μg/ml) DD for 2 h prior to addition of 13.2 μM (2 μg/ml) DD (blue).</p> <p>(B) Relative rate of NO production in pretreated (blue) and non-pretreated (green) cells. Experimental conditions as in (A).</p> <p>Representative data from five experiments is shown in (A). Data in (B) are means plus standard deviation from five experiments.</p> <p>a.u., arbitrary units.</p></div

DD Induces NO Generation in Diatoms

<div><p>Micrographs depicting NO generation over time in response to DD (66 μM [10 μg/ml]) in T. weissflogii (A) and P. tricornutum (B). (C) Monitoring of NO production in P. tricornutum in response to a range of DD concentrations; (D) Cytogram showing NO generation 15 min after addition of DEANO (2 mM) to P. tricornutum cells (filled violet indicates the KOH control; open green indicates DEANO). Insets show epifluorescence micrographs of the DEANO-treated cells. (E and F) Relative accumulation of NO in P. tricornutum cells following treatment with SNP (E) or NMMA prior to exposure to DD (F).</p> <p>In all experiments, NO generation was assayed using the fluorescent probe DAF-FM. Data in (C), (E) and (F) are means plus standard deviation from four experiments. Representative data from at least four experiments are shown in (A), (B), and (D). Experiments shown in (C), (D), and (F) were performed by flow cytometry, and in (E) using a fluorescence microplate reader.</p> <p>BF, bright field; Chl, chlorophyll-derived red autofluorescence; D, (2<i>E</i>)-decenal. Scale bars represent 5 μm.</p></div

Intercellular Transmission of a DD-Derived Signal that Induces NO in Neighboring Cells

<div><p>(A and B) In vivo imaging of DD-induced NO burst in P. tricornutum cells.</p> <p>(A) Time course of NO production in single cells based on corresponding real-time movie (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040060#sv001" target="_blank">Video S1</a>).</p> <p>(B) Relative accumulation of NO in selected cells of the micrographs shown in (A).</p> <p>(C) NO accumulation in an untreated population of DAF-FM-loaded cells that were inoculated with a non DAF-FM–loaded population that had been exposed to DD at different concentrations (open circle, methanol solvent; open square, 660 nM; filled circle, 3.3 μM; open triangle 6.6 μM, and open diamond, 13.2 μM) for 24 h prior to the mixing. Incubation of fresh medium with 13.2 μM DD for 24 h (filled triangle) prior to addition to DAF-FM-loaded cells did not provoke any detectable increase in NO. Data in (C) are means plus standard deviation from four experiments.</p></div

(A, B, C) Effect of the diatom PUA 2-<i>trans</i>,4-<i>trans</i>-decadienal (DD) on the human lung adenocarcinoma cell lines A549 and COLO 205, and the lung/brunch normal epithelial BEAS-2B cell line.

<p>(D) Effect of DD on A549 cell line in the presence of caspase-3 inhibitor (9.7 µM). Percentage of viable cells for A549 and COLO 205 calculated with the Trypan blue viability assay and for BEAS-2B with the MTT viability assay. Values are reported as mean ±S.D compared to controls (100% viability); ▴ 2 µM; ▪ 5 µM, ♦ 10 µM.</p

(A, B, C) Effect of the diatom PUA 2-<i>trans</i>,4-<i>trans</i>-heptadienal (HD) on the human lung adenocarcinoma cell lines A549 and COLO 205, and the lung/brunch normal epithelial BEAS-2B cell line.

<p>(D) Effect of HD on A549 cell line in the presence of caspase-3 inhibitor (9.7 µM). Percentage of viable cells for A549 and COLO 205 calculated with the Trypan blue viability assay and for BEAS-2B with the MTT viability assay. Values are reported as mean ±S.D compared to controls (100% viability); ▴ 2 µM; ▪ 5 µM, ♦ 10 µM.</p

Effect of the diatom PUAs 2-<i>trans</i>,4-<i>trans</i>-decadienal (DD), 2-<i>trans</i>,4-<i>trans</i>-octadienal (OD) and 2-<i>trans</i>,4-<i>trans</i>-heptadienal (HD) on the human lung adenocarcinoma cell line A549.

<p>Control and treated cells double stained with acridine orange and ethidium bromide after 48 µM DD, OD and HD observed at the confocal microscope. Numbers indicate (1) normal cells; (2) early apoptotic cells; (3) late apoptotic cells; (4) necrotic cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101220#s2" target="_blank">material and methods</a> for details). Arrows indicate cells with fragmented nuclei.</p

Schematic representation of pathways induced by 2-<i>trans</i>,4-<i>trans</i>-decadienal (DD), 2-<i>trans</i>,4-<i>trans</i>–octadienal (OD) and 2-<i>trans</i>,4-<i>trans</i>-heptadienal (HD) in A549 cell line.

<p>Black arrows indicate possible correlated pathways not involved in the response of cells to aldehyde treatment.</p