(G)SL and Sphk1 activity analysis in CD11b⁺ ATM isolated from ob/ob and lean mice SVF.

<p>A. Sphk1 activity. B. Sphingosine and C. S1P levels. Data depicted in the plots are analyzed using the Whitney-Mann test. D. Fold change (log2) values of GSL analyzed in WAT ob/ob vs lean CD11b⁺ macrophages are represented in a heat map (n = 4 and n = 6 in respective lean and ob/ob). The red color represents high-normalized levels and the blue color represents low levels of the lipids. Significance is evaluated by Whitney-Mann test and indicated; * p < 0.05; ** p < 0.01*** p < 0.001; † detected only in ob/ob CD11b⁺ cells and not included in the heat map analysis.</p

Sphk1 activity and S1P analysis in palmitate and LPS treated RAW264.7 cells.

<p>A. Sphk1 activity and B. S1P analysis normalized to respective controls. C. Fold change (log2) values of (G)SL analyzed in Raw264.7 challenged with BSA vs 500 μM BSA coupled to Palmitate (BSA-PA). The red color represents high-normalized levels and the blue color represents low levels of the lipids. (n = 3). Significance is evaluated by a student's t-test and indicated as * p < 0.05; ** p < 0.01 and ** p < 0.001.</p

Hierarchical cluster analysis (vertical) of expression of genes involved in (glyco)sphingolipid metabolism and transport in FACS sorted ATM populations.

<p>A. LFD ATMs (F4/80-Mgl1⁺[M2]) (n = 9 mice, 3 mice were pooled together for the analysis) and HFD ATMs (both F4/80-Mgl1⁺ [M2] and F4/80-Mgl1^- [M1]) (n = 6). The red color represents high-normalized expression levels and the blue color represents low expression levels. B. Relative <i>Sphk1</i> and C. <i>Sphk2</i> gene expression levels from LFD vs HFD ATM populations normalized by acidic ribosomal phosphoprotein 36B4 (P0). D. Hierarchical cluster analysis in WT, lean ATM (F4/80-Mgl1⁺[M2]) (n = 9 mice, 3 mice were pooled together for the analysis) and obese ob/ob ATM (both F4/80-Mgl1⁺ [M2] and F4/80-Mgl1^- [M1]) (n = 6 mice). E. Relative <i>Sphk1</i> and F. <i>Sphk2</i> gene expression levels lean vs. ob/ob relative to P0. Whitney-Mann test was used to evaluate significance in gene expression; * p < 0.05; *** p < 0.001.</p

<i>Sphk1</i> expression is induced by LPS, palmitate and chloroquine (CQ).

<p>A. <i>Sphk1</i> and B. <i>Sphk2</i> gene expression levels after 4h stimulation of RAW 264.7 with inflammatory mediators, hypoxia inducing CoCl2, ER stress inducing tunicamycin (TM), the lysosomal stressor CQ and 24 hours 500 μM BSA coupled palmitate (PA). Data are depicted as mean +/- S.D. (n = 3 per group). * p < 0.05; ** p < 0.01; *** p < 0.001.</p

Label-Free LC-MS^e in Tissue and Serum Reveals Protein Networks Underlying Differences between Benign and Malignant Serous Ovarian Tumors

<div><p>Purpose</p><p>To identify proteins and (molecular/biological) pathways associated with differences between benign and malignant epithelial ovarian tumors.</p><p>Experimental Procedures</p><p>Serum of six patients with a serous adenocarcinoma of the ovary was collected before treatment, with a control group consisting of six matched patients with a serous cystadenoma. In addition to the serum, homogeneous regions of cells exhibiting uniform histology were isolated from benign and cancerous tissue by laser microdissection. We subsequently employed label-free liquid chromatography tandem mass spectrometry (LC-MS^e) to identify proteins in these serum and tissues samples. Analyses of differential expression between samples were performed using Bioconductor packages and in-house scripts in the statistical software package R. Hierarchical clustering and pathway enrichment analyses were performed, as well as network enrichment and interactome analysis using MetaCore.</p><p>Results</p><p>In total, we identified 20 and 71 proteins that were significantly differentially expressed between benign and malignant serum and tissue samples, respectively. The differentially expressed protein sets in serum and tissue largely differed with only 2 proteins in common. MetaCore network analysis, however inferred GCR-alpha and Sp1 as common transcriptional regulators. Interactome analysis highlighted 14-3-3 zeta/delta, 14-3-3 beta/alpha, Alpha-actinin 4, HSP60, and PCBP1 as critical proteins in the tumor proteome signature based on their relative overconnectivity. The data have been deposited to the ProteomeXchange with identifier PXD001084.</p><p>Discussion</p><p>Our analysis identified proteins with both novel and previously known associations to ovarian cancer biology. Despite the small overlap between differentially expressed protein sets in serum and tissue, APOA1 and Serotransferrin were significantly lower expressed in both serum and cancer tissue samples, suggesting a tissue-derived effect in serum. Pathway and subsequent interactome analysis also highlighted common regulators in serum and tissue samples, suggesting a yet unknown role for PCBP1 in ovarian cancer pathophysiology.</p></div

Characterization of FACS sorted and CD11b⁺ isolated ATM phenotype.

<p>A. mRNA levels of the macrophage markers <i>Gpnmb</i>, B. <i>CD11c</i>, C. <i>FIZZ1</i> and D. <i>Mgl1</i> were analyzed by QPCR in CD11b⁺ enriched cells and compared to FACS sorted ATM from lean and ob/ob WAT. Whitney-Mann test was used to evaluate significance in gene expression; * p < 0.5; ** p < 0.05.</p

Sphk1 activity contributes to cell survival upon challenge of RAW264.7 cells with palmitate and chloroquine.

<p>The <i>CHOP/Sphk1</i> ratio is reduced in FACS sorted ATM A. HFD ATMs (both F4/80-Mgl1⁺ [M2] and F4/80-Mgl1^- [M1]) and B. ob/ob ATMs (both F4/80-Mgl1⁺ [M2] and F4/80-Mgl1^- [M1]). C. The <i>CHOP/Sphk1</i> ratio is also reduced in CD11b⁺ enriched cells from SVF D. S1P analysis showed that SK1-I inhibits Sphk1 activity. Analysis of the role of Sphk1 in promoting cell viability using E. WST-1 assay E. PARP cleavage analyzed by western blot in RAW264.7 cells challenged with 250 μM BSA coupled palmitate in the presence or absence of the Sphk1 inhibitor SK1-I for 24h (WST-1), or 8 h (PARP cleavage). Data in the graphs are depicted as mean +/- S.D. (n = 3) ** p < 0.01; *** p < 0.001.</p

Patients characteristics.

<p>Clinicopathological characteristics of the patient groups. SD: standard deviation.</p><p>Patients characteristics.</p

Venn diagram of the detected proteins.

<p>We detected a total of 84 proteins in serum and 209 in tissue, present in at least 50% of the samples in at least one of the conditions (benign or malignant). The grey area represents the proteins with an adjusted p-value of <0.05 when comparing benign with malignant.</p

PCBP1 expression in serous ovarian cancer.

<p>Expression of PCBP1 (probeset 208620_at) in serous tumors of low malignant potential (LMP) versus malignant serous ovarian tumors <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108046#pone.0108046-Anglesio1" target="_blank">[28]</a>. PCBP1 was significantly up-regulated (p = 0.003, Welch's t-test) in malignant tumors. Squares represent the individual samples used in the microarray experiment. Boxplots are overlaid with the lower and upper ends of a box indicating the 25th and 75th percentiles, respectively. The solid black line inside a box indicates the median.</p