RKIP expression in glioma cell lines.

<p><b>A</b>) Immunocytochemistry analysis of RKIP in glioma cell lines cells with both nuclear and cytoplasmic expression. <b>B</b>) Western blot analysis confirming the expression of RKIP at different levels in glioma cell lines. <b>C</b>) For RKIP inhibition, U251 cells were stably transfected with a shRNA for RKIP and with the respective empty vector for control. The band densitometry analysis showed that the shRKIP transfection induced a reduction of around 50% of the protein levels in relation to the control cells. Further, the cells were stimulated with 50 ng/ml of EGF by 10 minutes and ERK pathway activation was assessed by western blot for phospho-ERK1/2 expression. ERK pathway was overactivated in shRKIP transfected cells after EGF stimulation. Quantification of western blot results, using the band densitometry analysis, was performed with Image J software. For RKIP relative protein expression results are shown as the ratio between RKIP and β-Actin and for ERK activity the results are shown as the ratio between p-ERK1/2 and total ERK1/2.</p

<i>In vivo</i> role of RKIP in U251 cells growth and angiogenesis.

<p><b>A</b>) Representative pictures (16× magnification) of CAM assay after 7 days of tumor growth <i>in ovo</i> and <i>ex ovo</i>. <b>B</b>) Tumor growth was measured <i>in vivo</i> by CAM assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030769#s4" target="_blank">materials and methods</a> section. We observed a larger perimeter (µM) in the tumors formed by U251 shRKIP cells, there was no significant difference from control cells (upper panel). The counting of the blood vessels <i>ex ovo</i> revealed no differences in the number of vessels recruited in the tumors formed by shRKIP cells when compared to the control (lower panel). We analyzed 18 eggs (7 were injected with empty vector and 11 with shRKIP U251 transfected cells). The data is represented as the mean ± SD and differences with a <i>p</i><0.05 on the Student's t test were considered statistically significant.</p

<i>In vitro</i> role of RKIP in U251 cells biological behavior.

<p><b>A</b>) The cellular viability was measured at 24, 48 and 72 hours by MTS. RKIP inhibited cells had a viability advantage at 72 hours, when compared to control cells. <b>B</b>) Cell cycle analysis was done at 24 hour time point by flow cytometric analysis of propridium iodide stained cells. No differences were found in the cell cycle distribution. <b>C</b>) In the wound healing migration assay, a standardized scratch (wound) was applied to monolayers and digital images were taken at several time points (0, 24 and 72 hours). We observed that shRKIP transfected cells had a migration advantage at 24 and 72 hours. <b>D</b>) Representative images of the assay at 0 and 72 hours are represented (40× magnification). All the experiments were done in triplicate at least three times. Data is represented as the mean ± SD and differences with a <i>p</i><0.05 on the Student's t test were considered statistically significant (*).</p

RKIP expression in gliomas and associations with patients clinicopathological data (n = 193).

<p>N: Number of cases; SD: Standard deviation; WHO: World Health Organisation; p: person X² value; TMZ: Temozolomide; RT: Radiotherapy.</p

Immunohistochemistry analysis of RKIP in gliomas.

<p><b>A</b>) Normal brain and <b>B</b>) astrocytoma grade II showing high expression. <b>C</b>) Positive and <b>D</b>) Negative expression in oligodendroglioma grade II. <b>E</b>) Positive and <b>F</b>) Negative expression in glioblastoma. All the pictures were taken with at 200× magnification.</p

Independent prognostic factors in gliomas.

<p>HR: Hazard ratio, 95% CI: 95% Confidence interval.</p

The metabolic microenvironment of melanomas: Prognostic value of MCT1 and MCT4

<p><i>BRAF</i> mutations are known drivers of melanoma development and, recently, were also described as players in the Warburg effect, while this reprogramming of energy metabolism has been identified as a possible strategy for treating melanoma patients. Therefore, the aim of this work was to evaluate the expression and prognostic value of a panel of glycolytic metabolism-related proteins in a series of melanomas. The immunohistochemical expression of MCT1, MCT4, GLUT1, and CAIX was evaluated in 356 patients presenting melanoma and 20 patients presenting benign nevi. Samples included 20 benign nevi, 282 primary melanomas, 117 lymph node and 54 distant metastases samples. <i>BRAF</i> mutation was observed in 29/92 (31.5%) melanoma patients and 17/20 (85%) benign nevi samples. <i>NRAS</i> mutation was observed in 4/36 (11.1%) melanoma patients and 1/19 (5.3%) benign nevi samples. MCT4 and GLUT1 expression was significantly increased in metastatic samples, and MCT1, MCT4 and GLUT1 were significantly associated with poor prognostic variables. Importantly, MCT1 and MCT4 were associated with shorter overall survival. In conclusion, the present study brings new insights on metabolic aspects of melanoma, paving the way for the development of new-targeted therapies.</p

Immunophenotyping of paediatric glioma cell lines.

<p>All lines were grown as monolayers and stained with a variety of glial and stem cell markers including glial fibrillary acidic protein (GFAP), S100, vimentin, synaptophysin, nestin and CD133. H&E – haematoxylin and eosin. All images original magnification ×400.</p

Characteristics and immunophenotype of paediatric glioma cells grown as monolayers.

<p>The astrocytic nature of the cells was confirmed in culture by immunohistochemistry with a range of glial and stem cell markers. (+++) strongly positive; (++) moderately positive; (+) weakly positive; (−) negative.</p

Expression profiling of paediatric and adult glioblastoma cell lines.

<p>(A) Heatmap demonstrating hierarchical clustering of 93 differentially expressed genes between paediatric (SF188, KNS42, UW479) and adult (LN229, A172, U118MG, U87MG, SF268) high grade glioma cell lines. (B) Quantitative real-time (TaqMan) RT-PCR confirming differential expression of <i>CRKL</i>, <i>LYN</i>, <i>EPHA6</i> and <i>AXL</i>. Expression values are plotted relative to Universal Human Reference RNA. (C) Gene Set Enrichment Analysis highlighting co-ordinated differential expression of gene sets defined <i>a priori</i>. Enriched in paediatric high grade glioma cell lines - MORF_MSH2, GNF2_MLH1, GCM_RAD21, DNA_replication_reactome; enriched in adult lines IL6_SCAR_FIBRO_UP, CROONQUIST_IL6_RAS_UP, TGFBETA_C1_UP. Nominal p value<0.001.</p