Role of TNFalpha, IL-1beta and MIP-1alpha in HZ-dependent induction of lysozyme release from human monocytes.

<p>Cells were unfed (negative controls) or fed with HZ (positive controls) for 2 h. Afterwards, cells were incubated for 2 h alone and with a single dose (20 ng/ml) or a combination of rhTNFalpha, rhIL-1beta and rhMIP-1alpha (Panel A, mimicking approach); alternatively, cells were incubated for 2 h alone and with a single dose (30 ng/ml) or a combination of anti-hTNFalpha, anti-hIL-1beta or anti-hMIP-1alpha blocking antibodies (Panel B, blocking approach). Thereafter, lysozyme release was measured by spectrometric assay. Data are means + SEM of three independent experiments. Lysozyme release from monocytes is indicated as enzyme activity units measured in a 2 ml-well of cell supernatants. All data were evaluated for significance by ANOVA. Panel A: Vs unstimulated cells (column 1) *p<0.05, **p<0.01, ***p<0.0001; Vs untreated HZ-fed cells (column 2) °p<0.05, °°p<0.01. Panel B: Vs unstimulated cells (column 1) *p<0.0001; Vs untreated HZ-fed cells (column 2) °p<0.0001.</p

Involvement of p38 MAPK pathway in HZ-dependent induction of lysozyme release from human adherent monocytes.

<p>Cells were unfed (negative controls) or fed with HZ (positive controls) for 2 h; after phagocytosis, cells were incubated for 2 h alone and with 10 microM p38 MAPK synthetic inhibitor SB203580. Therefore, p38 MAPK protein expression and phosphorylation was evaluated by WB in cell lysates (Panels A and B), whereas lysozyme release was measured by spectrometric assay in cell supernatants (Panel C). Results are shown as a representative blot (A–B) or means + SEM (C) of three independent experiments. Lysozyme release from monocytes is indicated as enzyme activity units measured in a 2 ml-well of cell supernatants. In lysozyme release studies, data were also evaluated for significance by ANOVA. Vs unstimulated cells (column 1) *p<0.0001; Vs untreated HZ-fed cells (column 2) °p<0.0001.</p

Involvement of NF-kappaB pathway in HZ-dependent induction of lysozyme release from human adherent monocytes.

<p>Cells were unfed (negative controls) or fed with HZ (positive controls) for 2 h; after phagocytosis, cells were incubated for 2 h alone and with: 15 microM quercetin, an inhibitor of cytosolic I-kappaBalpha protein phosphorylation (Panels A–C); 10 microM artemisinin, an inhibitor of p65 and p50 NF-kappaB subunits nuclear translocation (Panels D–F); and 10 microM parthenolide, an inhibitor of DNA/NF-kappaB complex binding (Panels G–H). Therefore, I-kappaBalpha protein phosphorylation (A) and degradation (B), along with p65 (D) and p50 (E) nuclear translocation were evaluated by WB in cytosolic and nuclear fractions of cell lysates, respectively; DNA/NF-kappaB complex binding (G) was evaluated by EMSA in nuclear fraction of cell lysates; and lysozyme release was measured by spectrometric assay in cell supernatants (C, F, H). Results are shown as representative images (WB and EMSA studies) or means + SEM (lysozyme release studies) of three independent experiments. Lysozyme release from monocytes is indicated as enzyme activity units measured in a 2 ml-well of cell supernatants. In lysozyme release studies, data were also evaluated for significance by ANOVA. Panel C: Vs unstimulated cells (column 1) *p<0.0001; Vs untreated HZ-fed cells (column 2) °p<0.0001. Panel F: Vs unstimulated cells (column 1) *p<0.0001; Vs untreated HZ-fed cells (column 2) °p<0.0001. Panel H: Vs unstimulated cells (column 1) *p<0.0001; Vs untreated HZ-fed cells (column 2) °p<0.0001.</p

Early time-dependent induction of lysozyme release from HZ-fed human adherent monocytes: role of the lipid moiety of HZ.

<p>Cells were unfed (control cells, CTR) and fed with HZ, sHZ or dHZ for 2 h; then, lysozyme levels in cell supernatants were monitored 0, 1, 2, 24 and 48 h after the end of phagocytosis by spectrometric assay. Data are means + SEM of three independent experiments. Lysozyme release from monocytes is indicated as enzyme activity units measured in a 2 ml-well of cell supernatants. All data were evaluated for significance by ANOVA. Vs CTR *p<0.0001; Vs HZ °p<0.0001.</p

2-DE analysis of NB cells treated or not with ATRA.

<p>Representative image from three independent experiments of coomassie-stained 2-DE patterns of NB cell line SJ-N-KP: (<b>A</b>) 24 h control cells; (<b>B</b>) 24 h ATRA treatment; (<b>C</b>) 9 days control cells; (<b>D</b>) 9 days ATRA treatment. Proteins were separated using 17-cm, pH-4–7 strips followed by SDSPAGE on 10%, 18×20-cm gels. Proteins showing apparent differential expression were identified by progressive numbers on the <i>black squares</i>. Corresponding identifications are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018254#pone-0018254-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018254#pone-0018254-t002" target="_blank">2</a>.</p

Schematic representation of the proposed mechanism.

<p>AE1 is dynamically associated with the cytoskeleton through ankyrin binding in untreated red cells (A). In G6PD deficient RBCs diamide causes irreversible AE1 disulfide cross-linking and its phosphorylation by Syk kinase, diamide also causes progressive hemichrome formation. In control RBCs AE1 oxidation and phosphorylation are transient and no hemichrome formation is observed (B). Hemichromes bind to AE1 and promote the clustering of phosphorylated AE1 (C). Large aggregates of AE1 and hemichromes are released in vesicles (D).</p

Distribution of identified spots corresponding to proteins differentially phosphorylated following ATRA treatment.

<p>Distribution is shown according to proteins cellular compartment localization and their molecular function.</p

Proteins differentially expressed in neuroblastoma cell line following 9 days ATRA treatment as identified by MALDI-Tof MS.

a<p>Spot number were defined according to spot positions in 2-DE in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018254#pone-0018254-g002" target="_blank">Fig. 2</a>.</p>b<p>MW, molecular weight.</p>c<p>pI, isoelectric point.</p>d<p>Number of matched mass values on number of total mass values searched.</p>e<p>The sequence coverage, which is calculated as the percentage of identified sequence to the complete sequence of the matched protein.</p>f<p>Ratio between level of expression in treated and untreated cells. Standard deviation is indicated. For the significance two-sided Student's <i>t</i> test was used (* p<0.05, ** p<0.01).</p

Proteins differentially expressed in neuroblastoma cell line following 24 h ATRA treatment as identified by MALDI-Tof MS.

a<p>Spot number were defined according to spot positions in 2-DE in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018254#pone-0018254-g002" target="_blank">Fig. 2</a>.</p>b<p>MW, molecular weight.</p>c<p>pI, isoelectric point.</p>d<p>Number of matched mass values on number of total mass values searched.</p>e<p>The sequence coverage, which is calculated as the percentage of identified sequence to the complete sequence of the matched protein.</p>f<p>Ratio between level of expression in treated and untreated cells. Standard deviation is indicated. For the significance two-sided Student's <i>t</i> test was used (* p<0.05, ** p<0.01).</p

1-DE western blot analysis of NB cells treated or not with ATRA.

<p>Representative western blot (of three independent experiments) with anti-phosphoserine (<b>A</b>) and anti-phosphotyrosine (<b>B</b>) antibodies. SJ-N-KP cells were treated with ATRA 10 µM for 30, 60 minutes, 3, 9, 24, 48 hours and 9 days. Control cells (c) were not treated. 15 µg of proteins were analysed on 10% SDS-polyacrylamide gel. Western blot analysis with anti-actin antibodies was performed on the same membranes and used for data normalization. Data (means ± SD) expressed as densitometric units are shown in the lower corner of panels <b>A</b> and <b>B</b>. Significance of the differences between controls and ATRA treated cells: <i>p</i>≤0.05.</p