ATPase activity of liposome-reconstituted Pdr11p and Aus1p.

<p>Purified Pdr11p and Aus1p were reconstituted into different liposomes (containing Rho-PE as fluorescent lipid marker) and assayed for ATPase activity using [<i>γ</i>-³²P] ATP. A: SDS PAGE analysis of a flotation assay of Pdr11p proteoliposomes in a sucrose gradient. Detection of lipids and protein in the same low density fraction validated successful reconstitution. Proteins are visualised by silver staining and lipids by fluorescence from Rho-PE. B: Relative ATPase activity of Pdr11p reconstituted in PS liposomes in presence of the indicated inhibitors: orthovanadate, 1 mM; BeSO₄, 1 mM; NaF, 5 mM. Data is based on at least two reconstitutions from one purification batch. C: Lipid effect on ATPase activity of reconstituted Pdr11p and Aus1p. All activities are corrected for protein amount in the proteoliposomes. Data is based on two reconstitutions from one purification batch of each protein. PC, PC only; PS, PC/PS (1:1); PG, PC/PG (7:3).</p

Overexpression and functionality of tagged Pdr11p variants.

<p><i>S. cerevisiae</i> cells were transformed with empty vector (e.v.) or pESC-URA carrying <i>PDR11</i>, <i>PDR11-GFP</i>, or <i>PDR11^K788M</i>. Sizes of the molecular mass standards are given to the left when relevant. A: Western blot illustrating the induced expression of FLAG-tagged variants of Pdr11p (filled arrowheads) and breakdown product (open arrowhead). Cells carrying the empty vector served as control. The blot was stained with anti-FLAG antibody. B: In-gel fluorescence detection using Criterion TGX stain-free gel. C: Serial dilutions of transformed heme-deficient <i>hem1</i>Δ and sterol uptake-deficient triple mutant <i>hem1</i>Δ<i>aus1</i>Δ<i>pdr11</i>Δ strains spotted onto standard synthetic galactose plates supplemented with <i>δ</i>-aminolevulinic acid (ALA) or cholesterol.</p

Solubilisation and purification of Pdr11p from <i>hem1</i>Δ<i>aus1</i>Δ<i>pdr11</i>Δ cells.

<p>A: Flowchart of the procedure. Supernatant S1 was centrifuged at 10,000 or 60,000 g (45 min) for collection of plasma membrane enriched fractions or total yeast membranes, respectively. B: Solubilisation of Pdr11p from total yeast membranes. Equal amounts of membranes (P2) were solubilised at the given DDM concentrations. After ultracentrifugation, the amounts of solubilised and non-solubilised Pdr11p were estimated in aliquots of supernatants (S3) and pellets (P3), respectively, via immunoblotting with anti-FLAG antibodies. Pdr11p, filled arrowheads; breakdown product, open arrowhead. C, original membrane fraction not been subjected to solubilisation (positive control). C: Purification of Pdr11p from plasma membrane enriched fractions. Representative Western blot analysis with anti-FLAG antibodies and Coomassie stained SDS-PAGE (SDS) of selected purification fractions. The loaded purification fractions are normalised with respect to volume. Sizes of the molecular mass standards are indicated on the sides.</p

Expression analysis utilising GFP-tagged Pdr11p.

<p>A: Effect of induction time on Pdr11p-GFP expression in <i>hem1</i>Δ<i>aus1</i>Δ<i>pdr11</i>Δ. Panels I-IV, Representative flow cytometry based histograms of cells transformed with plasmids carrying <i>PDR11-GFP</i> (green) or empty vector (black) after 6 and 17 h in SD medium (non-induced) or SG medium (induced). Each data set consists of minimum 20,000 cells. Panels V-VIII, Representative fluorescence microscopy images of cells expressing Pdr11p-GFP after 6 and 17 h induction. The intensities of the fluorescent signals cannot be compared between the images. Bars equal 10 <i>μ</i>m. B: Percentage of cells expressing Pdr11p-GFP after galactose induction as a function of optical density at 600 nm (OD₆₀₀) and cell concentration of the glucose pre-culture just prior to media change. All cultures in each repetition grew from a single colony. Lines are included solely to guide the eye. <i>hem1</i>Δ<i>aus1</i>Δ<i>pdr11</i>Δ strain, expression after 6 h induction in 3 cultures from the same selected high-expressing colony. BJ1991 strain, expression after 8 (open triangles) and 10 h (open diamonds) from the same selected colony.</p

ATPase activity of solubilised Pdr11p.

<p>ATPase activity of the purified detergent-solubilised transporter was assayed as described under “Materials and Methods” using [<i>γ</i>-³²P] ATP. A: ATPase activity as a function of pH. Open and filled circles are data from two independent experiments. Values are normalised with respect to the values at pH 7.2 (open circles) or pH 7.4 (closed circles). The dashed line is included to guide the eye. B: Effect of various inhibitors: NaN₃, 5 mM; ouabain, 5 mM; BeSO₄, 1 mM; NaF, 5 mM; AlF₃, 1 mM; orthovanadate, 1 mM; EDTA, 1 mM. C: ATPase activity as a function of orthovanadate concentration. Fitting of data to a dose-response/activity curve (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184236#sec002" target="_blank">Material and methods</a>) gives <i>IC</i>₅₀ = 4 ± 2 mM, and a Hill coefficient = 0.8 ± 0.2. Results in B and C are the mean ± S.D. from at least two independent experiments relative to the value obtained for the purified detergent-solubilised protein in the absence of inhibitors (control).</p

Effect of various compounds on ATPase activity of purified Pdr11p^a.

<p>Effect of various compounds on ATPase activity of purified Pdr11p<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184236#t002fn001" target="_blank">^a</a>.</p

Conserved ABC motifs of Aus1p and Pdr11p.

<p>Conserved ABC motifs of Aus1p and Pdr11p.</p

Levels of intracellular metabolite pools in aerosol-exposed macrophages.

<p>Pools of (a) lactic acid, (b) succinic acid, and itaconic acid were measured. Levels represent the mean of 9 biological replicates. *** = p < 0.001. Error bars represent s.e.m.</p

HFO particles induce activation of immune response in RAW 264.7 macrophages.

<p>(a) The Gene Ontology term GO:0006955, corresponding to activation of immune response, was found to be significantly up-regulated in HFO-treated samples (p = 0.059) and not regulated in the DF-treated samples. (b) Model of how the regulated proteins found in this study affect the NF-kB immune response pathway in the cell. Stimulation of the toll-like receptor (TLR2) leads to activation of NF-kB. Tumor necrosis factor alpha-induced protein 8-like protein 2 (TNFAIP8L2) acts as a negative regulator of TLR2, preventing hyperresponsiveness of the immune system, and inhibiting NF-kappa-B activation. Peroxiredoxin 2 (Pdrx2) reduces hydrogen peroxide, inhibiting NF-kappa-B activation.</p

Experimental set-up and global omics analyses.

<p>(A) An 80 KW common-rail-ship diesel engine was operated with heavy fuel oil (HFO) or refined diesel fuel (DF). The exhaust aerosols were diluted and cooled with clean air. On-line real-time mass spectrometry, particle-sizing, sensor IR-spectrometry and other techniques were used to characterise the chemical composition and physical properties of the particles and gas phase. Filter sampling of the particulate matter (PM) was performed to further characterise the PM composition. Lung cells were synchronously exposed at the air-liquid-interface (ALI) to aerosol or particle-filtered aerosol as a reference. The cellular responses were characterised in triplicate at the transcriptome (BEAS-2B), proteome and metabolome (A549) levels with stable isotope labelling (SILAC and ¹³C₆-glucose). (B) Heatmap showing the global regulation of the transcriptome, proteome and metabolome.</p