MET analysis in ERL-resistant cell lines.

<p>A) qPCR analysis of gene copy numbers of <i>MET;</i> B) western blots of total cell lysates with the antibodies indicated of parental (P) and ERL-resistant cell lines; C) qPCR analysis of MET mRNA expression in parental (P) and ERL-resistant cell lines. <i>MET</i> gene and mRNA in A) and C) are normalized to RNaseP gene and rp-L31 mRNA respectively and both are expressed relative to the levels in parental cell lines set as 1 (mean ± SD of triplicate determinations). qPCR data are representative of those obtained from 2 independent analysis; D) Confocal microscopy analysis of MET receptor (green) expression in xenograft nodes of mice subcutaneously injected with the parental HCC827 cells and the ERL-resistant RA1, RB1 and RA2 cell lines. Images show representative xy-plane maximum projection of the specimens. Scale bars correspond to 15 μm.</p

Exogenous HIPK2 expression is not sufficient to induce apoptosis or sensitizes MNSC cells to DNA damaging drugs. A

<p>, HIPK2 knock-down was achieved by transient transfection with sh-RNAi in MNA IMR-5 cells and was measured by Q-RT-PCR (inset). Analysis of bleomycin (5µg/ml) induced apoptosis is shown as percentage of apoptotic nuclei and cells positive for p85 cleaved fragment of the PARP protein (p85^PARP). Significant differences in apoptosis fold induction were obtained between HIPK2i and CTRi transfected cells (***p<0.0001) <b>B</b>, Cell transfection with HIPK2 (+) but not empty vector (−) caused apoptosis in the U2OS osteosarcoma cells as indicated by the appearance of apoptotic nuclei and/or positive staining for p85^PARP (left panel), but failed to induce apoptosis and to sensitize MNSC SHEP neuroblastoma cells to bleomycin treatment (percentage of the apoptotic cells are given in the graphs in the right panel). The immunoblot (lower right panel) shows the accumulation of the indicated proteins in HIPK2 transfected and/or bleomycin treated cell extracts.</p

HER2/HER3 and AXL expression and phosphorylation analysis.

<p>A) Representative western blots of total cell lysates of HCC827 and HCC4006 parental cell lines (P) and their derived ERL-resistant cell lines. Arrows indicate the expected molecular weight size. Total cell lysates loaded were 40 μg for AXL and pAXL analyses and 25 μg for the others. B) qPCR analysis of AXL mRNA normalized to rp-L31 mRNA and expressed relative to the levels in parental cell lines set as 1 (mean ± SD of triplicate determinations). Western blots and qPCR data are representative of those obtained respectively from 3 and 2 independent analysis. C) Dose-effect curves were calculated using CompuSyn software and plotting the entered Fa values against the entered dose values. For combination treatments, the combined drugs dose was entered. Each data point represents the mean of 3 replicates.</p

Analysis of <i>EGFR</i> and <i>KRAS</i> gene mutations.

<p>Analysis of <i>EGFR</i> and <i>KRAS</i> gene mutations.</p

Analysis of the <i>EGFR</i> gene in the RA2 ERL-resistant cell line.

<p>A) Analysis of <i>EGFR</i> exon 19 nucleotides sequence. The pherogram of the parental cell line with peaks corresponding to the <i>EGFR</i> mutated sequence (ΔE746-A750) and the pherogram of the RA2 resistant cells with peaks corresponding to the mutated and wild type (WT) EGFR nucleotides sequence are shown. B) qPCR analysis. Relative <i>EGFR</i> gene copy number (GCN) in genomic DNA, normalized to the <i>Rnase P</i> gene, is expressed relative to the levels in parental cell lines (P) set as 1 (mean ± SD of triplicate determinations). Results are representative of those obtained from 2 independent analysis.</p

HIPK2 overexpression and Galectin-3 knock-down cooperate in sensitizing MNSC cells to apoptosis. A

<p>, Percentage of apoptotic nuclei in MYCN expressing SHEP cells following Gal-3 overexpression (immunoblot, lower panel) and bleomycin treatment. Significant differences in apoptosis fold induction were obtained between Gal-3 and pcDNA (CTR) transfected cells (**p<0.01). <b>B</b>, Gal-3 knock-down was achieved via transient transfection with RNAi duplexes in MNSC SHEP cells (inset). Analysis of bleomycin induced apoptosis is shown as percentage of apoptotic nuclei (upper panel) and p85^PARP positive cells (lower panel). Significant differences in apoptosis fold induction were obtained between Gal-3i and CTRi transfected cells (***p<0.0001). <b>C</b>, HIPK2 overexpression and Gal-3 knock-down were obtained in MNSC SHEP cells (inset) and the effect on apoptosis in basal condition and following bleomycin treatment was analyzed by counting the percentage of apoptotic nuclei (upper panel) and p85^PARP positive cells (lower panel). Significant differences in apoptosis fold induction were obtained in Gal-3i+HIPK2 transfected cells compared either to CTR or to Gal3i transfected cells (*p≤0.05; **p<0.01).</p

Dm₅₀ of single agent and drugs combination for parental and ERL-resistant NSCLC cell lines.

<p>Dm₅₀ of single agent and drugs combination for parental and ERL-resistant NSCLC cell lines.</p

MYCN regulates Galectin-3 expression. A

<p>, Average Gal-3 mRNA expression (+/− standard deviation) in MNSC and MNA NB tumor samples and NB cell lines measured by Q-RT-PCR (***p<0.0001). Raw data are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049139#pone.0049139.s001" target="_blank">Fig. S1</a>. <b>B</b>, Immunoblot of Gal-3 protein expression in MNSC and MNA NB cells. <b>C</b>, Immunoblot (lower panels) and Q-RT-PCR analysis (upper panels) of Gal-3 expression in SHEP Tet21/N MYCN inducible cells and SK-N-MYC compared to parental SK-N-SH cells (***p<0.0001). <b>D</b>, MYCN knock-down was achieved via transient transfection with RNAi duplexes in MNA LAN1 cells and the expression of the indicated proteins was investigated by immunoblot.</p

Nutlin-3 efficiently induces cell death and cooperates with clastogenic drugs by downregulating Gal-3 in MNA NB cells.

<p><b>A</b>, Cell death induced by either cis-platin (CDDP, 1 µM), Adriamycin (ADR, 0.1 µM), Nut-3 (2 or 10 µM) or combination of Nut-3 (2 µM) with CDDP or ADR, was measured by Tripan blue-exclusion test (upper panel) in the indicated MNA NB cell lines and the expression of the indicated proteins was assessed by immunoblot (lower panel). Significant differences in cell death were obtained between Nut-3 treated samples versus the corresponding untreated controls (CTR) and/or single drug treated samples (**p<0.01; ***p<0.0001). B, Effect of Gal-3 overexpression on apoptosis induced by ADR and Nut-3 in IMR32 cells. Data are represented as averages (+/− standard deviations) of the apoptosis fold induction compared to untreated controls. Significant differences in apoptosis fold induction were obtained between Gal-3 and CTR transfected cells (**p<0.01; ***p<0.0001). C, Effects of Nut-3 (16 µM) on bleomycin induced apoptosis and Gal-3 expression in MNSC SHEP cells. Significant differences in apoptosis fold induction were obtained between bleomycin and Nut-3 treated samples versus bleomycin-only treated samples (***p<0.0001).</p

Galectin-3 intracellular localization in NB cells.

<p>To address Gal-3 localization, immunofluorescent analysis was performed on fixed MNSC (<b>A</b>) and MNA (<b>B</b>) NB cell lines; for each cell type Gal-3 immunostaining alone (left panels) and double staining with the MitoTracker (right panels) are shown. <b>C</b>, Immunoblot analysis of Gal-3 content in cell equivalent amounts of mitochondrial (M) and cytosolic (C) extracts from the indicated NB cell lines; p38^MAPK and Cyt-c were used as controls for cytosolic and mitochondrial enrichment, respectively. <b>D</b>, Effect of Gal-3 repression by RNAi (inset) on bleomycin-induced apoptosis in IMR32 NB cells shown as percentage of apoptotic nuclei and p85^PARP positive cells. Significant differences in apoptosis fold induction were obtained between Gal-3i and CTRi transfected cells (*p≤0.05; **p<0.01).</p