Supplementary Figure S2 from The IL-18 Antagonist IL-18–Binding Protein Is Produced in the Human Ovarian Cancer Microenvironment

Supplementary Figure S2 - PDF file 2513K, A and B: Analysis of the correlation between IL-18 and IL-18BP protein levels in EOC sera (n. 47) (A) and ascites (n. 17) (B). No significant correlation (P=ns) was found by Pearson's test. C: Analysis of the correlation between IL18 and IL-18BP protein levels and IFN-? in the ascites of 15 EOC patients. IFN-? levels are low to undetectable and show no correlation with IL-18 or IL-18BP levels (P=ns). Pearson's correlation coefficients are shown (r). Lines represent the best-fit linear regression analysis with the 95% Confidence Interval</p

Supplementary Figure S6 from The IL-18 Antagonist IL-18–Binding Protein Is Produced in the Human Ovarian Cancer Microenvironment

Supplementary Figure S6 - PDF file 185K, A: Analysis of the correlation between IL18BP and EBI3 mRNA levels in high grade (Type II) tumors in two microarray datasets of EOC. A significant correlation was found between EBI3 and IL18BP in both datasets, suggesting a relationship between the expression of EBI3 and IL18BP mRNA in EOC cell primary tumors. Pearson's correlation coefficients are shown (r). Lines represent the best fit linear regression analysis with the 95% Confidence Interval. B: Association between different EBI3 mRNA expression levels and Progression Free Survival (PFS) in Type II EOC of the Tothill microarray dataset. High EBI3 mRNA levels are associated to a shorter PFS time. Median PFS was 13 months for EBI3 levels higher than third quartile versus 21 months for EBI3 levels lower than first quartile (P=0.016). Solid line: cases with EBI3 levels lower than first quartile (n.52). Dashed line: cases with EBI3 levels higher than third quartile (n.49). P values were determined using log-rank test.</p

Supplementary Figure S3 from The IL-18 Antagonist IL-18–Binding Protein Is Produced in the Human Ovarian Cancer Microenvironment

Supplementary Figure S3 - PDF file 9497K, A: Immunochemical analysis of IL-18BP expression in cells from EOC ascites. Double staining with anti-IL-18BP (red, rabbit mAb clone EP1088Y, Epitomics) and anti-macrophage (brown, mAb HAM-56, Ventana Medical Systems) antibodies is shown. Biotin-labeled goat anti-rabbit followed by alkaline phosphatase-conjugated streptavidin and peroxidase-conjugated anti-mouse (BioSpa) were used as secondary antibodies. Fast Red and DAB (Sigma) served as substrates. Bar=100�m. Some of the cells stained by the anti-macrophage Ab were also stained by anti-IL-18BP Ab (see enlarged inset). However, the brightest IL-18BP positive cells were negative for the anti-macrophage Ab. B: Two-color immunofluorescence analysis of IL-18BP protein versus leukocyte surface markers expression in cells from EOC ascites. Numbers indicate the % of cells in each quadrant. IL-18BP positive cells were CD13 positive and CD14 low or negative (as all the CD14 positive cells were within the CD13 positive population: lower right panel). FITC: fluorescein isothiocyanate; APC: allophycocyanin; PE: phycoerythrin. CD14APC and CD14PE were from Miltenyi Biotec, CD13PE from BD Pharmingen and NKp46PE from Beckman Coulter. Cells were analyzed on a FACSCalibur (Becton Dickinson) flow cytometer</p

Supplementary Figure S4 from The IL-18 Antagonist IL-18–Binding Protein Is Produced in the Human Ovarian Cancer Microenvironment

Supplementary Figure S4 - PDF file 640K, Immunochemical analysis of IL-18BP expression in EOC cell lines and xenotransplants. Immunochemistry with an anti-IL-18BP Ab shows virtually no reactivity in EOC cell lines (A upper left panel, B left panel), whereas IL-27-cultured A2774 cells (B middle panel) and areas of orthotopic SKOV3 (A) and A2774 (B right panel) xenotransplants express IL-18BP. The sections were observed with a Nikon Eclipse 80i light microscope equipped with a color camera imaging head, using a 40x objective. Bar=100micron</p

Changes in ALCAM expression induced by ionomycin or phorbol myristate acetate (PMA).

<p>Total lysates (Lys) and conditioned medium (CM) from TPC-1 cells, treated or not with 1 µM ionomycin (iono) or 50 ng/mL PMA for 2 h, were resolved by 3–8% SDS-PAGE and immunoblotted with ALCAM antibody. Both treatments led to an increase of secreted ALCAM isoforms; especially PMA induced a gain of 60-kDa isoform expression. Arrow indicates the membrane-localized ALCAM isoform in lysate, and arrowheads indicate shed-ALCAM isoforms in CM, respectively. Beta-actin was used as a loading control.</p

ALCAM expression in thyroid cell lines.

<p>Analysis of TT and MZ-CRC1 cell lysates showing ALCAM protein expression in these thyroid cell lines. SKOV-3 and TPC-1 cell lysates were included as controls. Total lysates from each cell line were resolved by 4–12% SDS-PAGE and immunoblotted with anti-ALCAM antibodies. Arrow indicates the fully glycosylated ALCAM isoform. Beta-actin was used as a loading control.</p

ALCAM expression in normal and tumor human thyroid tissue samples.

<p>(A) Total protein extracts (30 µg) from 11 papillary thyroid carcinomas (Ca Pap), 5 medullary thyroid carcinomas (Ca Mid), and 5 normal thyroid tissues (Thyroid) were resolved by 4–12% SDS-PAGE, transferred onto a nitrocellulose membrane, and immunoblotted with anti-ALCAM antibody. (*) indicates a non-specific background band. Beta-actin was used as a loading control. (B) Western blot analysis of ALCAM expression of total protein extracts (30 µg) from three PTCs, two MTCs, and three CTRLs, treated (+) or not (−) with N-glycosidase F. Beta-actin was used as a loading control.</p

Supplementary Figure S1 from The IL-18 Antagonist IL-18–Binding Protein Is Produced in the Human Ovarian Cancer Microenvironment

Supplementary Figure S1 - PDF file 744K, Receiver Operating Characteristic (ROC) curve for IL-18BP serum levels at diagnosis in patients with all types and stages of ovarian tumors (malignant n. 48 versus normal controls n. 13): the area under the curve (AUC) value is significant with the 95% confidence interval indicated in parentheses. SE=standard error</p

Supplementary Data from z-Leucinyl-Leucinyl-Norleucinal Induces Apoptosis of Human Glioblastoma Tumor–Initiating Cells by Proteasome Inhibition and Mitotic Arrest Response

Supplementary Figures S1-S6 and Supplementary Tables S1-S3.</p

Release of ALCAM is sensitive to ADAM17/TACE silencing.

<p>(A) Analysis of lysates from TPC-1 and A2774 cells, resolved on 4–12% SDS-PAGE and immunoblotted with anti-ADAM17/TACE antibodies. Arrows indicate inactive (130 kDa) and active (80 kDa) forms of ADAM17/TACE enzyme. Normalization of results was obtained with immunoblotting analysis of beta-actin. (B) Expression of ADAM17/TACE protein by TPC-1 cells transfected with an ADAM17/TACE-specific small interfering RNA (siRNA) (OTP17), or with non-targeting siRNA (NT) as detected by western blot. The amount of protein was calculated by comparative densitometric scanning with beta-actin. (C) ELISA detection of ALCAM release by TPC-1 cells after transfection with siRNA specifically inhibiting ADAM-17/TACE (OTP17, black column) or with non-targeting siRNA (NT, white column). (D) Conditioned medium (CM) from TPC-1 cells, cultured with pervanadate (PV) (60 min), epidermal growth factor (EGF) (24 h), or medium alone (ctr, 24 h) in the presence (black columns) or in absence (white columns) of CGS27023A (CGS) was assessed by ELISA for ALCAM. Columns, means of three experiments (cells cultured in presence of 10, 1, or 0.1 µM CGS); bars, SD. *, P<0.05. Grey bar: in absence of CGS, but in presence of orthovanadate (OV).</p