Cofactor expression in HNSCC cell lines.

<p>The expression of p300 <b>(a)</b>, PGC1α <b>(b)</b>, SRC1 <b>(c)</b>, SRC2 <b>(d)</b> and SRC3 <b>(e)</b> as nuclear receptor coactivators, RXRα <b>(f)</b> as PXR heterodimerization partner and NCoR1 <b>(g)</b>, NCoR2 <b>(h)</b> and SHP <b>(i)</b> as nuclear receptor corepressors was assessed at mRNA level in a set of 8 HNSCC cell lines through qRT-PCR. The expression of the different target genes was normalized to <i>RNA Polymerase II (RPII)</i> expression, used as housekeeping gene. Data are expressed as mean ± S.D. (n = 3). To allow for comparisons between cell lines, results were normalized to the expression in HNO97 cells, set to 1.</p

Caspase activity in NCoR2 over-expressing HNO97, HNO124 and HNO210 cells.

<p>Cells were transfected with pCMV6-NCoR2 plasmid (or the empty vector pCMV6). Caspase activity was assessed at 24 h post-transfection in HNO97 <b>(a)</b>, HNO124 <b>(b)</b> and HNO210 <b>(c)</b> cells. Results were normalized to the corresponding activity in cells transfected with the pCMV6 empty vector, set to 1. *significantly different from pCMV6-transfected cells, p<0.05, n = 3. As a positive control, cells were treated with staurosporine (1 μM, 24 h), a known inductor of apoptosis. Subsequently, caspase activity was assessed in HNO97 <b>(d)</b>, HNO124 <b>(e)</b> and HNO210 <b>(f)</b> cells. Results were normalized to the corresponding measurements in control cells (vehicle treated), set to 1. Data are expressed as mean ± S.D. *significantly different from control cells, p<0.05, n = 3.</p

Cleaved caspase 3 and cleaved PARP expression in NCoR2 over-expressing HNO97, HNO124 and HNO210 cells.

<p>Cells were transfected with pCMV6-NCoR2 plasmid (or the empty vector pCMV6). Cleaved caspase 3 and cleaved PARP protein levels were evaluated at 24 h post-transfection in HNO97 <b>(a)</b>, HNO124 <b>(b)</b> and HNO210 <b>(c)</b> cells through western blot. β-actin was used as a loading control. Representative western blot detections are shown for each cell line. *significantly different from pCMV6-transfected cells, p<0.05, n = 3.</p

Correlation between coactivator- and corepressor expression and PXR transcriptional activity.

<p>Correlation between coactivator- and corepressor expression and PXR transcriptional activity.</p

mRNA expression of drug transporters in HNSCC.

<p>(A) Determination of mRNA expression levels of drug transporters by qRT-PCR in a study sample of therapy naïve stage IVa HNSCC tumors (n = 40, grey boxes) and normal control samples (n = 14, white boxes) (relative mRNA expression normalized to the lowest value). (B) External validation by HNSCC RNAseq mRNA expression data derived from the ‘The Cancer Genome Atlas’ (TCGA) consortium. Comparison of normalized counts of paired tumor (n = 37, grey boxes) and adjacent noncancerous normal tissue (n = 37, white boxes). Whisker indicates 5–95 percentile; Mann-Whitney U test; **, p<0.01; ***, p<0.001.</p

Additional file 4: of Chronophin regulates active vitamin B6 levels and transcriptomic features of glioblastoma cell lines cultured under non-adherent, serum-free conditions

Figure S3. Analysis of phosphocofilin levels in CIN knockdown cell lines. (A) Representative example of a western blot after PhosTag gel electrophoresis probed with α-cofilin antibody. (B) and (C) Quantification of western blots as shown in (A) for NCH644 (B) and NCH421k (C). There is no significant difference in the P-cofilin/total cofilin ratio between CTRL or CIN shRNA cells (n = 3, one-way ANOVA followed by Dunnett’s multiple comparisons test, p > 0.05). Shown are means + SD. (TIF 328 kb

Correlation of low mRNA expression and shortened survival of HNSCC patients.

<p>Classification of HNSCC patients according to the gene expression level in the high (n = 20) or low (n = 20) expression group. Survival analysis by Kaplan-Meier curves and log rank test revealed a significant correlation of lower expression and shortened progression-free and overall survival.</p

Additional file 5: of Chronophin regulates active vitamin B6 levels and transcriptomic features of glioblastoma cell lines cultured under non-adherent, serum-free conditions

Figure S4. Quality control plots for next generation sequencing data. (A) and (C) There is a high enrichment of small p-values after standard filtering in both NCH644 (A) and NCH421k (C) as calculated by DESeq2. (B) and (D) MA-plots indicate the presence of many deregulated genes (red) in both NCH644 (B) and NCH421k (D). (TIF 1008 kb

Immunohistochemical staining of ATP7b protein expression in HNSCC.

<p>Immunohistochemical staining of ATP7b protein expression in HNSCC patients (n = 61) revealed a high range of immunoreactive scores (left). Grouping of patients according to their immunoreactive scores (cutoff  = 15) into low (n = 41) or high (n = 18) showed a trend towards a longer progression-free and overall survival in patients with a higher ATP7b expression (right).</p

Comprehensive Analysis of Glycolytic Enzymes as Therapeutic Targets in the Treatment of Glioblastoma

<div><p>Major efforts have been put in anti-angiogenic treatment for glioblastoma (GBM), an aggressive and highly vascularized brain tumor with dismal prognosis. However clinical outcome with anti-angiogenic agents has been disappointing and tumors quickly develop escape mechanisms. In preclinical GBM models we have recently shown that bevacizumab, a blocking antibody against vascular endothelial growth factor, induces hypoxia in treated tumors, which is accompanied by increased glycolytic activity and tumor invasiveness. Genome-wide transcriptomic analysis of patient derived GBM cells including stem cell lines revealed a strong up-regulation of glycolysis-related genes in response to severe hypoxia. We therefore investigated the importance of glycolytic enzymes in GBM adaptation and survival under hypoxia, both in vitro and in vivo. We found that shRNA-mediated attenuation of glycolytic enzyme expression interfered with GBM growth under normoxic and hypoxic conditions in all cellular models. Using intracranial GBM xenografts we identified seven glycolytic genes whose knockdown led to a dramatic survival benefit in mice. The most drastic effect was observed for <i>PFKP</i> (PFK1, +21.8%) and <i>PDK1</i> (+20.9%), followed by <i>PGAM1</i> and <i>ENO1</i> (+14.5% each), <i>HK2</i> (+11.8%), <i>ALDOA</i> (+10.9%) and <i>ENO2</i> (+7.2%). The increase in mouse survival after genetic interference was confirmed using chemical inhibition of PFK1 with clotrimazole. We thus provide a comprehensive analysis on the importance of the glycolytic pathway for GBM growth in vivo and propose PFK1 and PDK1 as the most promising therapeutic targets to address the metabolic escape mechanisms of GBM.</p></div