Additional file 4: Figure S3. of IGF1R activation and the in vitro antiproliferative efficacy of IGF1R inhibitor are inversely correlated with IGFBP5 expression in bladder cancer

IGF1R expression by epithelial cells in normal urothelium and bladder tumors. (a)Anti-IGF1R immunohistochemistry from human protein atlas project ( http://www.proteinatlas.org/ ). 3 examples of representative staining in tumors are presented in the right panel, staining of the two normal samples are presented in the left panel. Scale bar represents 100 μm (b) Haematoxylin-eosin staining of our CIT-series of tumors. Examples of tumors with high and low IGF1R expression assessed by RPPA. (TIFF 7100 kb

Additional file 3: Figure S2. of IGF1R activation and the in vitro antiproliferative efficacy of IGF1R inhibitor are inversely correlated with IGFBP5 expression in bladder cancer

IGF1R mRNA levels according to tumor stages in two independent publicly available data sets. (TIFF 7830 kb

Additional file 1: Table S1. of IGF1R activation and the in vitro antiproliferative efficacy of IGF1R inhibitor are inversely correlated with IGFBP5 expression in bladder cancer

mRNA expression levels of the components of IGF pathway in the FBLAD-Exon set of 125 bladder tumors. Data were obtained from Human Genome Exon 1.0ST arrays. (XLS 27 kb

Several deregulated genes have their expression correlated with the expression of <i>MKI67</i>, a proliferation marker gene.

<p>The Pearson correlation coefficient (r) between the expression of the deregulated genes and the expression of proliferation marker gene, <i>MKI67</i>, was calculated for <i>FGFR3</i>-mutated tumors in the TaG1G2 group (n = 28) and for <i>FGFR3</i>-non-mutated tumors in the T2–4 group (n = 63). The expression of the deregulated genes as a function of <i>MKI67</i> expression is shown in TaG1G2 <i>FGFR3</i>-mutated tumors (upper figures) and in T2–4 non-mutated tumors (lower figures). Only the plots for the correlated genes are presented (p<1%, which corresponds to a correlation coefficient, |r| above 0.479 for the <i>FGFR3</i>-mutated tumor group and above 0.323 for the <i>FGFR3</i>-non-mutated tumor group).</p

Rab and Rab-interacting proteins.

<p>Example of the Rab27 cluster. The Rab27 cluster is comprised of the two <i>RAB27</i> isoforms (<i>RAB27A</i> and <i>RAB27B</i>), the GEF <i>MADD</i>, the GAP <i>TBC1D10A</i> and 12 effector proteins. The other Rab and Rab-interacting proteins are shown in supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039469#pone.0039469.s001" target="_blank">Figure S1</a>.</p

Deregulated genes correlated with differentiation markers.

<p>The Pearson correlation coefficient (r) of the expression of the deregulated genes with the expression of urothelial differentiation markers in <i>FGFR3</i>-mutated superficial tumors (TaG1G2) (n = 28 samples) and <i>FGFR3</i>-non-mutated muscle-invasive tumors (T2–4) (n = 63) is presented. Correlation with p<1% (|r| above 0.479 for Ta-T1 tumors, and |r| above 0.323 for <i>FGFR3</i>-non-mutated T2–4 tumors) are written in bold.</p

Results obtained after the different analysis steps.

<p>The results of each analysis step are shown in the flow chart presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039469#pone-0039469-g001" target="_blank">Figure 1</a>.</p

Expression in normal samples and in the two groups of tumors (mutated and non-mutated for <i>FGFR3</i>) classified according to stage, of the genes found to be specifically deregulated in one of the two pathways of bladder tumor pathogenesis.

<p>Expression of <i>SYTL1</i>, <i>LEPRE1</i>, <i>MICAL2</i>, <i>RAB23</i> and <i>STXBP1</i> measured by Affymetrix array in normal samples (n = 4) and <i>FGFR3</i>-mutated tumor samples (TaG1G2, n = 28; T1, n = 13; T2-4, n = 9), and <i>FGFR3-</i>non-mutated samples (TaG3, n = 3; T1, n = 25; and T2-4, n = 63). Are represented the 10th percentile (below bar), the 25th percentile (box bottom), the median (bar in the box), the 75^th percentile (box top) and the 90^th percentile (upper bar). The points represent the outlier samples. <i>SYTL1</i> is down-regulated in <i>FGFR3</i>-non-mutated tumors, whereas <i>LEPRE1</i>, <i>MICAL2</i>, <i>RAB23</i> and <i>STXBP1</i> are up-regulated.</p

Deregulated genes during bladder cancer pathogenesis in the <i>FGFR3</i>-non-mutated tumor pathway.

<p>From line 1 to line 19, genes are down-regulated. From line 20 to line 31, genes are up-regulated. The results in bold passed the thresholds: FC<0.667 (for down-regulation) or >1.5 (for up-regulation) and q-value <5%.</p><p>FC: Fold Change.</p><p>Number of FGFR3-non-mutated tumor samples: 3 TaG3, 25 T1, 63 T2–4.</p><p>Number of normal urothelial samples: 4.</p

Up-regulated gene expression in normal human urothelium (NHU) cells and bladder tumor cell lines.

<p>The expression of the 13 up-regulated genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039469#pone-0039469-t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039469#pone-0039469-t003" target="_blank">3</a>) (<i>CAV1</i>, <i>ITGA5</i>, <i>KIF20A</i>, <i>LEPRE1</i>, <i>MICAL1</i>, <i>MICAL2</i>, <i>RAB23</i>, <i>RAB31</i>, <i>RABAC1</i>, <i>SDC1</i>, <i>STXBP1</i>, <i>TMEM22</i> and <i>ZWINT</i>) was measured by Affymetrix array in 7 bladder cancer cell lines (KK47, MGHU3, RT112, RT4, SCaBER, SD48 and T24) and normal human urothelial (NHU) cells grown in culture. The threshold for genes to be considered as up-regulated in tumor cell lines (2 fold the expression measured in NHU cells) is represented by a black line.</p