MOESM1 of Low Percentage of KRAS Mutations Revealed by Locked Nucleic Acid Polymerase Chain Reaction: Implications for Treatment of Metastatic Colorectal Cancer

Low Percentage of KRAS Mutations Revealed by Locked Nucleic Acid Polymerase Chain Reaction: Implications for Treatment of Metastatic Colorectal Cance

Supplementary Table 2 from Role of CXCL13-CXCR5 Crosstalk Between Malignant Neuroblastoma Cells and Schwannian Stromal Cells in Neuroblastic Tumors

Supplementary Table 2 from Role of CXCL13-CXCR5 Crosstalk Between Malignant Neuroblastoma Cells and Schwannian Stromal Cells in Neuroblastic Tumor

Supplementary Table 1 from Role of CXCL13-CXCR5 Crosstalk Between Malignant Neuroblastoma Cells and Schwannian Stromal Cells in Neuroblastic Tumors

Supplementary Table 1 from Role of CXCL13-CXCR5 Crosstalk Between Malignant Neuroblastoma Cells and Schwannian Stromal Cells in Neuroblastic Tumor

Clinicopathological Characteristics of 70 Patients with Lung Adenocarcinoma and IL-12Rβ2 Expression Profiles of their Tumors

*<p>IL-12Rβ2 immunostaining was scored as negative, positive, weakly positive or mixed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006119#s4" target="_blank">methods</a>.</p><p>ND: not detected.</p><p>ADBF: adenocarcinomas with bronchioloalveolar features.</p><p>BAC: bronchioloalveolar carcinomas.</p><p>ADC: adenocarcinomas.</p

IL-12Rβ2 expression and function in normal bronchial epithelial cells.

<p>4A. IL-12RB2 expression in human primary bronchial epithelial cells, as assessed by RT-PCR. From left to right: MW = molecular weight; NC = negative control (Raji cell line); three different NBEC cultures (NBEC #1, #2 and #3) are shown. 4B. IL-12Rβ2 surface expression in human NBEC, as assessed by flow cytometry. Open profile: IL-12Rβ2 staining; dark profile: isotype matched mAb staining. 4C. Short circuit current recordings in normal bronchial epithelial cells. The figure depicts two representative experiments from control (<i>top</i>) and IL-12 treated (<i>bottom</i>) epithelia showing responses to amiloride (10 µM, apical), forskolin (20 µM, apical and basolateral), UTP (100 µM, apical), and CFTR_inh-172 (10 µM, apical). 4D. Cytokine release by human NBEC, as assessed by Bio-Plex Assay. Pooled results from supernatants of three different bronchial epithelial cell suspensions are shown. IL-12 treatment reduced significantly the release of IL-6 (P = 0.0049), IL-8 (P = 0.0071), FGF-b (P = 0.0231), GM-CSF (P = 0.0028), IP-10 (P<0.0001), MCP-1 (P = 0.0002) and RANTES (P = 0.0108).</p

IL-12Rβ2 expression and function in human NSCLC cell lines.

<p>2A. IL-12RB2 expression in NSCLC cell lines, as assessed by RT-PCR. From left to right: MW = molecular weight; NC = negative control (Raji cell line); PC = positive control (total tonsil B cells); different NSCLC cell lines (Colo699, Calu6, Calu1, A549, SK-MES-1, GLC82 and Calu6/β2 cells) are shown. 2B. Left panel. IL-12Rβ2 protein expression in Calu6/β2 cells, as assessed by flow cytometry. Open profile: IL-12Rβ2 staining; dark profile: isotype matched antibody staining. Right panel. IL-6 intracellular staining in Calu6/β2 cells cultured with medium or hrIL-12 for 48 h, as assessed by flow cytometry. Open profile: IL-6 staining in cells cultured with medium; dark profile: isotype matched antibody staining, dashed line: IL-6 staining in cells cultured with IL-12. 2C. Angiogenic activity of supernatants from Calu6/β2 cells cultured with medium alone or hrIL12. CAM treated with sponges loaded with supernatant from the untreated cells were surrounded by allantoic vessels developing radially towards the implant in a ‘spoked-wheel’ pattern (upper left panel). When supernatants from hrIL-12treated Calu6/β2 cells was tested, a significant reduction (P = 0.001) of the angiogenic response was appreciable (upper right panel). Lower panels show the angiogenic activity of Calu6/β2 cells in the presence of an anti-IL-6 mAb (left panel) or of an anti-VEGF-C mAb (right panel). These experiments were repeated three times. Original magnification: ×50.</p

Anti-tumor activity of IL-12 on NSCLC <i>in vivo.</i>

<p>3A. Volume of tumors grown after Calu6/β2 cell inoculation orthotopically (left panel) or subcutaneously (right panel) in PBS and hrIL-12 treated animals. Animals injected orthotopically were sacrificed after 23 days, those injected subcutaneously after 14 days. Volume of tumors grown after inoculation orthotopically (left panel) or subcutaneously (right panel) of Calu6 cell transfected with the empty vector hrIL-12 treated animals was also shown (empty vector+IL12). The differences in size between tumors removed from PBS and hrIL-12 treated mice were evaluated by Mann-Whitney U test. Boxes indicate values between the 25^th and 75^th percentiles, whisker lines represent highest and lowest values for each group. Horizontal lines represent median values. 3B. Tumors (developed after s.c injection) injected subcutaneously with Calu6/β2 cells in PBS-treated SCID/NOD mice are mostly formed of nests of undifferentiated, pleomorphic and proliferating cells (mitotic (figures) features indicated by arrows) rapidly infiltrating the underlying muscle layers (arrowheads) (a), and supplied by a distinct microvessel network, as assessed by laminin staining (b). In hrIL-12 treated mice, tumor histology is altered by the appearance of large areas of ischemic-hemorrhagic necrosis (N) (c) associated with defective microvascular architecture (d) (×400). Orthotopical injection of Calu6/β2 cells gave rise, in PBS-treated mice, to tumors with istopathological features (e) similar to those of subcutaneously developed tumors (a) and supplied by a well developed microvascular network (f). As observed in subcutaneous tumors, in orthotopic tumors as well hrIL-12 treatment induced wide necrosis (g) and severe microvascular alterations (h) (×400). 3C. Human Angiogenesis PCR Array on tumors explanted from hrIL-12 <i>vs</i> PBS treated animals 23 days after orthotopic inoculation of Calu6/β2 cells. Histogram shows fold expression changes of genes in tumors from hrIL-12 <i>vs</i> PBC treated mice. 3D. Tumors from PBS-treated mice express VEGF-C (a) and IL-6 (c). Expression of VEGF-C and IL-6 is strongly reduced (b and d, respectively) in tumors from hrIL-12 treated mice. (×400).</p

IL-12Rβ2 expression and function in human lung adenocarcinoma.

<p>1A. Histological features and IL-12Rβ2 expression in human bronchioloalveolar lung carcinomas. Non-mucinous bronchioloalveolar carcinoma typically shows columnar neoplastic cells growing along the alveolar septa (a). In 41.4% of adenocarcinomas, neoplastic cells lack IL12Rβ2 expression (b), though a few may sometimes retain it (inset in b). By contrast, alveoli unaffected by malignant process express IL-12Rβ2 (c), as observed in the remaining tumors (d). (×400). 1B. Angiogenic activity of supernatants from one representative lung ADC sample cultured in the presence or absence of hrIL12. CAM treated with sponges loaded with supernatant from the untreated cells were surrounded by allantoic vessels developing radially towards the implant in a ‘spoked-wheel’ pattern (left panel). When supernatants from hrIL-12 treated lung ADC sample was tested, a significant reduction (P = 0.001) of the angiogenic response was appreciable (right panel). These experiments were repeated three times. Original magnification: ×50. 1C. Pooled results from human angiogenesis PCR array performed in three lung ADC samples cultured in the presence or absence of hrIL-12 are shown. Histogram shows fold expression changes of genes in primary samples treated with hrIL-12 vs medium.</p

Additional file 1 of Expression of Immunoglobulin Receptors with Distinctive Features Indicating Antigen Selection by Marginal Zone B Cells from Human Spleen

Supplementary material, approximately 2.13 MB

Analysis of Mda-9/syntenin protein expression in uveal melanoma cell lines.

<p><b>A:</b> Immunostaining of fixed and permeabilized cell lines (left) and primary cultures (right). Original magnification 400×. The insets show the negative control performed by the use of non-immune rabbit Ig.</p