Isoflurane releases CD73 containing microparticles in cultured endothelial cells.

<p>A. Human umbilical vein endothelial (EA.hy926) cells were treated with 0–2.5% isoflurane for 1 hr and endothelial cell culture media microparticles (MP) were isolated and assayed for CD73 activity. Isoflurane caused a significant increase in human endothelial cell microparticle CD73 activity (N = 5–8). B. Microparticles isolated from mouse glomerular endothelial cells (GENC) treated with 2.5% isoflurane for 1 hr also had higher CD73 activity compared to carrier gas-treated cells (N = 4–6). C and D. Representative CD73 immunoblotting images (C) and band intensity quantifications (D) from microparticles isolated from EA.hy926 cells. Beta-actin protein expression was also quantified to normalize lane loading. Isoflurane treatment (2.5% for 1 hr) significantly increased CD73 protein expression in EA.hy926 cell microparticles compared to carrier gas-treated cells. *P<0.05 vs. carrier gas group. Error bars represent 1 SEM.</p

Primers used to amplify cDNAs based on published GenBank sequences for human.

<p>bp, base pairs; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ICAM-1, intercellular adhesion molecule-1; TNF-α, tumor necrosis factor-alpha; VCAM-1, vascular cell adhesion molecule-1; CD73, Ecto-5′-nucleotidase. Respective anticipated PCR product size (bp, base pairs), PCR cycle number for linear amplification, and annealing temperatures used for each primer are also provided.</p

Flow cytometric analyses of endothelial cell culture media microparticles.

<p>A. Representative flow cytometric analyses of microparticles isolated from EA.hy926 endothelial cell culture media. EA.hy926 cells were treated with 2.5% isoflurane or with carrier gas for 1 hr and isolated microparticles were incubated with CD73 antibody and Annexin V. B. EA.hy926 cells treated with 2.5% isoflurane for 1 hr had significantly higher CD73+ Annexin V+ microparticles compared to microparticles isolated from carrier gas-treated endothelial cells (N = 5). *P<0.05 vs. carrier gas group. Error bars represent 1 SEM.</p

Isoflurane increases adenosine generation in cultured endothelial cells.

<p>Adenosine in media from human umbilical vein endothelial cells (EA.hy926, A) or mouse glomerular endothelial cells (GENC, B) measured with high pressure liquid chromatography. Isoflurane treatment (0–2.5%) for 6 hr increased adenosine concentrations when compared to carrier gas-treated controls (N = 5). Data are presented as means ± SEM. *P<0.05 vs. carrier gas-treated controls. Error bars represent 1 SEM.</p

Isoflurane stimulates Rho kinase activity in human endothelial cells.

<p>A. Rho kinase activity in human endothelial (EA.hy9262) cells was measured by detecting myosin phosphatase target protein-1 phosphorylation after treatment with 2.5% isoflurane or with carrier gas for 30 min. Isoflurane significantly increased Rho kinase activity in EA.hy9262 cells compared to carrier gas-treated cells (N = 5). *P<0.05 vs. carrier gas group. Error bars represent 1 SEM. B. EA.hy9262 endothelial Rho kinase activity was also assessed by detecting myosin light chain (MLC) phosphorylation in EA.hy9262 cells with immunoblotting. MLC phosphorylation increased in EA.hy9262 cells treated with 2.5% isoflurane for 30 min compared to carrier gas-treated cells. Total MLC immunoreactivity did not change with isoflurane treatment. Representative of 2 experiments performed in triplicate.</p

Isoflurane transiently increases CD73 activity without changing CD73 synthesis in cultured endothelial cells.

<p>A. Human umbilical vein endothelial (EA.hy926) cells treated with 2.5% isoflurane showed a significant but transient induction of CD73 activity. CD73 activity peaked at 3 hr and then decreased to near baseline at 6–16 hr after isoflurane treatment (N = 6–8). B. Isoflurane treatment for 3 hr caused dose-dependent increase in CD73 activity in EA.hy926 cells compared to carrier gas-treated cells (N = 4–5). Data are presented as means ± SEM. *P<0.05 vs. CD73 activity measured at baseline (A) or in cells treated with 0% isoflurane (B). C and D. Representative images for CD73 mRNA (RT-PCR) and protein (immunoblotting) expression in EA.hy926 cells. EA.hy926 cells were treated with carrier gas or with 2.5% isoflurane for 6 hr (C) or for 16 hr (D). Isoflurane treatment did not increase CD73 mRNA or protein expression in EA.hy926 cells. Representative of 3–4 experiments.</p

Endogenous tagging and generation of knockouts in <i>T</i>. <i>gondii</i>.

<p><b>A</b>. Schematic of the CRISPR/Cas9 tagging system. Tagging plasmids were generated with various tags (green box) flanked by common ends (red and black boxes) and including a common stop codon (gray box) followed by the <i>HXGPRT</i> 3’ UTR (yellow box) and the selectable marker HXGPRT. Amplification of this central region with primers that contained short homology regions HR1 (purple box) and HR2 (blue box) together with the common flanks (red and black boxes) generated products for gene-specific tagging. Co-transfection of these amplicons with a CRISPR/Cas9 plasmid bearing the gene-specific single guide RNA (sgRNA3’) was used to add an epitope tag (green box) at the C-terminus of the endogenous locus. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006379#ppat.1006379.s006" target="_blank">S1 Fig</a> for more details. <b>B</b>. Localization of CaM1, CaM2 and CaM3 containing C-terminal 6HA tags. Detected with mouse anti-HA (green) and rabbit anti-GAP45 (red). Scale bar, 2 μM. <b>C</b>. Schematic of the double CRISPR/Cas9 gRNA system used for generation of clean knockouts using two sgRNAs matching the 5’ and 3’ ends of the coding sequence. The entire coding sequence was replaced by the DHFR marker flanked by short homology regions (HR3, red; HR2, blue). Primers (p) used for diagnostic PCR. <b>D</b>. Diagnostic PCR of knockouts compared to the parental ku80^KO line. <i>CDPK1</i>, PCR control. <b>E.</b> Plaque numbers formed by the knockouts compared to the parental ku80^KO line. ns, not significant, analyzed by one-way ANOVA.</p

Generation of AID tagged lines in the TIR parental line of <i>T</i>. <i>gondii</i>.

<p><b>A.</b> Western blot analysis using antibodies to detect CaM1-AID or CaM3-AID (mouse anti-HA to the AID-3HA tag), TIR1-3Flag (rat anti-Flag) and aldolase (rabbit anti-aldolase, ALD). <b>B and C.</b> Degradation of AID tagged proteins in cam2^KO<i>/</i>CaM1-AID (<b>B</b>) and CaM3-AID (<b>C</b>) lines after addition of auxin (500 μM IAA) for different time periods. Mock indicates parasites grown with 0.1% ethanol for 36 hr. CaM1-AID or CaM3-AID proteins were detected with mouse anti-HA and rabbit anti-aldolase (ALD) antibodies served as a loading control. Band intensities were analyzed by ImageJ, and ratios of anti-HA vs. anti-ALD signal were calculated (HA/ALD) and expressed as a percentage of the mock treatment (i.e. 100%). <b>D and E</b>. Degradation of AID tagged proteins in cam2^KO<i>/</i>CaM1-AID (D) and CaM3-AID (E) parasites after 24 hr incubation with 500 μM IAA (+IAA) or ethanol vehicle 0.1% (-IAA). CaM1-AID or CaM3-AID proteins were detected with mouse anti-HA (green) and rabbit GAP45 (red) antibodies served as a control to label the parasite. Scale bar, 2 μM. <b>F</b>. Plaque formation by parasites grown on HFF monolayers. Scale bar, 0.5 cm. Insert images in the CaM3-AID line, scale bar (red) = 1 mm. <b>G</b>. Measurement of plaque numbers and sizes for the CaM3-AID line treated with and without auxin. N≥ 25, ***, <i>P</i> < 0.0001. Mann Whitney non-parametric test.</p

Development of the auxin inducible degron (AID) system in <i>T</i>. <i>gondii</i>.

<p><b>A.</b> Structure of auxin indole-3-acetic acid (IAA). <b>B</b>. Plaque formation of the ku80^KO line grown in 500 μM IAA or ethanol control (0.1%) (mock) for 6 days. Scale bar = 2 mm. <b>C.</b> Plaque formation of <i>the</i> ku80^KO line grown in IAA or ethanol control (0.1%) mock) for 6 days. Mean ±S.D. from three independent experiments with triplicates for each (n = 9). ns, not significant, analyzed by one-way ANOVA. <b>D.</b> Heterologous co-expression of <i>Oryza savita</i> auxin receptor TIR1 (TIR1-3xFLAG)(parental line) (red) and an N-terminal YFP fusion with the <i>Arabidopsis thaliana</i> IAA17 (AID) (YFP-AID-3xHA) (green) in the TIR1 background. Scale bar, 2 μm. <b>E.</b> Schematic illustration of conditional degradation of AID-tagged proteins in <i>T</i>. <i>gondii</i>. <b>F.</b> Dose-response and <b>G.</b> time course of YFP-AID-3HA knockdown following IAA treatment by Western blotting with antibodies to YFP-AID-3HA (mouse anti-HA) or Aldolase (as a loading control).</p

Phenotypic comparison among <i>T</i>. <i>gondii</i> mutant lines.

<p>Phenotypic comparison among <i>T</i>. <i>gondii</i> mutant lines.</p