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

TrkAIII attenuation of mitochondrial ROS production is reversed by TrkA inhibitors.

<p>A) Digital fluorescence micrographs demonstrating Mitosox-reactive ROS (red) in untreated pcDNA, TrkA and TrkAIII SH-SY5Y cell lines (Control) or following treatment for 12 hours, at 37°C with: 1 µM Rotenone; 250 µM Paraquat; or for 3 hours at 37°C with 1 µM LY83583 (bar = 100 µm). B) Histograms showing densitometric fold difference in Mitosox reactive ROS levels in pcDNA, TrkA and TrkAIII SH-SY5Y cells following 12 hours treatment with 1 µM Rotenone (Rot); for 12 hours with 250 µM Paraquat (Par); or for 3 hours with 1 µM LY83583, compared to untreated controls (Con), given the arbitrary value of 1±s.d. Results are displayed as mean±s.d fold difference compared to untreated controls in three independent experiments. Mean values are provided above each column and asterisks denote statistical significance by t-test comparison of treated cell lines to untreated controls. C) Digital fluorescence micrographs demonstrating Mitosox-reactive ROS (red) in untreated TrkAIII SH-SY5Y cells (Control); TrkAIII SH-SY5Y cells treated for 36 hours with 1 µM CEP-701, 1 µM K252a, 1 µM Gö6976 or 100 nM GW441756, alone; TrkAIII SH-SY5Y cells treated for 12 hours with either 1 µM Rotenone, 250 µM Paraquat or for 3 hours with 1 µM LY83583, alone; and TrkAIII SH-SY5Y cells pre-incubated for 24 hours with either 1 µM Cep-701, 1 µM K262a, 1 µM Gö6976 or 100 nM GW441756, then treated for 12 hours with either 1 µM Rotenone, 250 µM Paraquat or for 3 hours with 1 µM LY83583, in the continuous presence of each respective inhibitor (bar = 100 µm). D) Histogram demonstrating the differences in ROS levels detected in untreated TrkAIII SH-SY5Y cells (Con); TrkAIII SH-SY5Y cells treated with 1 µM CEP-701 (CEP), 1 µM K252a (K252), 1 µM Gö6976 (Gö69), 100 nM GW441756 (GW4), 1 µM Rotenone (Rot), 250 µM Paraquat (Par) or 1 µM LY83583 (LY83), alone; or with 1 µM Rotenone (Rot), 250 µM Paraquat (Par) or 1 µM LY83583 (LY83), following pre-incubation with, and in the continuous presence of, 1 µM CEP-701, 1 µM K252a, 1 µM Gö6976 or 100 nM GW441756, as described for 5C. Results are expressed as mean±s.d densitometric units per 100 cells, in three independent experiments performed in duplicate. Mean values are provided above each column and asterisks denote statistical significance by Student’s t-test (p<0.0004, df = 10) and One-Way ANOVA ((2, 15) P<0.0001), comparing inhibitor alone, ROS-inducer alone and inhibitor plus ROS-inducer groups.</p

TrkAIII promotion of SH-SY5Y spheroid growth is inhibited by GW441756 and associates with stem cell marker expression.

<p>A) Representative digital phase-contrast micrographs, demonstrating differences in the relative size of 20-day pcDNA, TrkA and TrkAIII SH-SY5Y tumour spheroids (bar = 100 µm), plus a histogram showing the difference in 20-day pcDNA, TrkA and TrkAIII SH-SY5Y tumour spheroid volumes displayed as the mean+s.d. volume (mm³) of 50 individual tumour spheres per cell line, in three independent experiments. Mean values are provided above each histogram column and asterisks denote statistical significance by t-test comparison of TrkAIII SH-SY5Y cells to either pcDNA or TrkA SH-SY5Y cells. B) Digital phase-contrast micrographs demonstrating differences in the relative tumour spheroid sizes in TrkA and TrkAIII SH-SY5Y cells cultured for 20 days in the absence (Control) or presence of 100 nM or 1 µM GW441756 (bar = 100 µm), plus a histogram showing the difference in TrkA and TrkAIII SH-SY5Y tumour spheroid volumes, cultured as described above, displayed as the mean+s.d. volume (mm³) of 50 individual tumour spheres per cell line, in three independent experiments. Mean values are provided above each histogram column and asterisks denote statistical significance by t-test comparison of GW441756-treated and untreated TrkAIII SH-SY5Y cells. C) Ethidium bromide stained agarose gels demonstrating the relative levels of 4.2 kb SOD2, 1.5 kb SOD2 and GAPDH RT-PCR products in 20-day pcDNA, TrkA and TrkAIII SH-SY5Y spheroids, plus a histogram depicting the relative densitometric differences in 4.2 kb SOD2 and 1.5 kb SOD2 mRNA expression. Results are displayed as mean+s.d. densitometric ratio to GAPDH, in three independent experiments. Mean values are provided above each histogram column and asterisks denote statistical significance by t-test comparison of TrkAIII to both pcDNA and TrkA SH-SY5Y cells. D) Ethidium bromide stained agarose gels demonstrating differences in CD133, CD117, SOX-2, Nestin, Nanog and GAPDH RT-PCR products in 20-day pcDNA, TrkA and TrkAIII SH-SY5Y spheroids, plus histograms depicting fold change in (I), CD117, (II) SOX-2, (III) Nestin, (IV) Nanog and (V) CD133 expression in 20-day tumour spheroids. Results are displayed as mean±s.d. fold change compared to pcDNA SH-SY5Y cells, given the arbitrary value of 1 as a densitometric ratio to GAPDH expression, in three independent experiments. Mean values are provided above each histogram column and asterisks denote statistical significance by t-test comparison of TrkAIII to pcDNA SH-SY5Y cells.</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

SOD2 expression in TrkAIII SH-SY5Y cells is reduced by TrkA, IP3K and NF-κB inhibitors.

<p>A) Western blots demonstrating relative differences in TrkA, TrkAIII, phosphorylated TrkA (pY674/675-TrkA), SOD2, Trx-2 and α-tubulin levels in equal protein concentrations (20 µg) of total extracts from TrkA and TrkAIII SH-SY5Y cell lines, treated without (Con) or with 1 µM CEP-701 (CEP), 1 µM K252a (K2); 25 µM LY294002 (LY294) or 1 µM Gö6976 (Gö69) for 24 hours, at 37°C. B) Representative Western blots demonstrating relative differences in SOD2 and α-tubulin levels in equal protein concentrations (20 µg) of total extracts from TrkA and TrkAIII SH-SY5Y cell lines treated without (Con) or with: 100 nM GW441756 (GW44), 500 µM PDTC (PDTC), 100 µM Bay 11–7082 (BAY11) or 30 µM PD098059 (PD098). C) Representative Western blot of total cell extracts (20 µg), demonstrating inhibition of constitutive TrkAIII Y674/675 phosphorylation (Con) following 3 hours treatment of TrkAIII transfectants with 100 nM GW441756 (GW44). D) Histograms showing the densitometric differences in the SOD2 and Trx-2 levels, as a ratio to α-tubulin, in equal concentrations (20 µg) of total cell extracts from TrkA and TrkAIII SH-SY5Y cells treated without (Con) or with: 1 µM CEP-701 (CEP); 1 µM K252a (K2); 25 µM LY294002 (LY294); 1 µM Gö6976 (Gö69); 100 nM GW441756 (GW44); 500 µM PDTC (PDTC); 100 µM Bay 11–7082 (BAY11) or 30 µM PD098059 (PD098), for 24 hours at 37°C. Results are displayed as mean±s.d fold change compared to untreated controls, adjusted to the arbitrary value of 1±s.d., in 3 independent experiments. Mean values are presented above each column and asterisks denote statistical significance by t-test comparison of treated and untreated TrkAIII SH-SY5Y cells. E) Representative Western blots demonstrating differences in TrkAIII, phosphorylated TrkAIII (pTrkAIII), SOD2 and α-tubulin levels in equal concentrations (20 µg) of total cell extracts from wt-TrkAIII and kd-TrkAIII SH-SY5Y cell lines plus a histogram depicting the densitometric difference in the SOD2 levels as a ratio to α-tubulin, in total cell extracts from wt-TrkAIII and kd-TrkAIII SH-SY5Y cells, displayed as mean±s.d fold difference in densitometric ratio with respect to wt-TrkAIII transfectants adjusted to the arbitrary value of 1±s.d., in three independent experiments. Mean values are presented above each column and asterisks denote statistical significance by t-test comparison of wt and kd TrkAIII transfectants. F) Representative Western blots demonstrating levels of TrkA, tyrosine phosphorylated TrkA (pTrkA), SOD2 and α-tubulin in equal concentrations (20 µg) of total cell extracts from untreated TrkA SH-SY5Y cells (Con) and TrkA SH-SY5Y cells treated for 30, 60 and 180 minutes with 100 ng/ml NGF, plus a histogram showing densitometric differences in the SOD2: α-tubulin ratio in total cell extracts from untreated (Con) and NGF-treated TrkA SH-SY5Y cells (100 ng/ml for 30, 60 and 180 minutes), displayed as mean±s.d. fold difference in SOD2 levels with respect to untreated controls adjusted to the arbitrary value of 1±s.d., in 3 independent experiments. Mean values are provided above each column and asterisks denote statistical significance by t-test comparison between NGF-treated and untreated TrkA SH-SY5Y controls.</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

TrkAIII Augments Mitochondrial SOD-2 levels and Activity.

<p>A) Representative Western blots demonstrating relative SOD2, Trx2 and Hsp60 levels in equal protein concentrations (20 µg) of purified mitochondrial extracts from selected pcDNA, TrkA and TrkAIII SH-SY5Y cell lines. B) Histogram depicting the differences in SOD2 and Trx-2 levels, as a densitometric ratio to Hsp60, in pcDNA, TrkA and TrkAIII SH-SY5Y cell lines. Results are displayed as mean±s.d fold difference in densitometric ratio with respect to pcDNA transfectant controls adjusted to the arbitrary value of 1±s.d., in three independent experiments. C) Representative SOD activity zymogram demonstrating relative levels of SOD1 and SOD2 activity in equal protein concentrations (150 µg) of purified mitochondrial extracts from selected pcDNA, TrkA and TrkAIII SH-SY5Y cell lines. D) Histogram depicting differences in SOD2 activity levels as a densitometric ratio to SOD1 activity levels. Results are displayed as the mean±s.d fold difference in densitometric ratio with respect to pcDNA transfectant controls, adjusted to the arbitrary value of 1±s.d., in 3 independent experiments performed in duplicate. For histograms, mean values are provided above each column and asterisks denote statistical significance by t-test comparison of TrkAIII SH-SY5Y cells to either pcDNA or TrkA SH-SY5Y cells.</p

TrkAIII protects SH-SY5Y cells against ROS-mediated death.

<p>A) Digital fluorescence micrographs demonstrating changes in viability (green = alive; red = dead) in pcDNA, TrkA and TrkAIII SH-SY5Y cells under basal conditions (Control) or treated for 24 hours at 37°C with either 1 µM Rotenone; 250 µM Paraquat; or for 6 hours with 1 µM LY83583, at 37°C (bar = 100 µm). B) Histograms showing differences in the viability of: (I) pcDNA; (II) TrkAI; and (III) TrkAIII SH-SY5Y cells, treated as described above. Results are displayed as mean±s.d percent viability, in three independent experiments performed in duplicate. Mean values are provided above each column and asterisks denote statistical significance by Student’s t-test comparison of Ros-inducer treated and untreated controls. C) Digital fluorescence micrographs demonstrating viability levels (green = alive; red = dead) in untreated TrkAIII SH-SY5Y cells (Control); TrkAIII SH-SY5Y cells treated with either 1 µM CEP-701, 1 µM K252a, 1 µM Gö6976, 100 nM GW441756, 1 µM Rotenone, 250 µM Paraquat or 1 µM LY83583 alone; or pre-incubated with either 1 µM Cep-701, 1 µM K262a, 1 µM Gö6976 or 100 nM GW441756, then treated with either 1 µM Rotenone, 250 µM Paraquat or 1 µM LY83583 in the continuous presence of each respective inhibitor (Cep-701, K262a, Gö6976 or GW441756), (bar = 100 µm). D) Histogram demonstrating differences in TrkAIII SH-SY5Y cell viability in untreated TrkAIII SH-SY5Y cells (Control); TrkAIII SH-SY5Y cells treated with either 1 µM CEP-701, 1 µM K252a, 1 µM Gö6976, 100 nM GW441756, 1 µM Rotenone, 250 µM Paraquat or 1 µM LY83583 alone; or pre-incubated with either 1 µM Cep-701, 1 µM K262a, 1 µM Gö6976 or 100 nM GW441756, then treated with either 1 µM Rotenone, 250 µM Paraquat or 1 µM LY83583 in the continuous presence of each respective inhibitor, as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094568#pone-0094568-g009" target="_blank">Fig. 9C</a>. Results are displayed as mean±s.d percent viability, in three independent experiments performed in duplicate. Mean values are provided above each column and asterisks denote statistical significance by Student’s t-test (p<0.0001, df = 10) and One-Way ANOVA ((2, 15) p<0.0001), comparing inhibitor alone, ROS-inducer alone and inhibitor plus Ros-inducer groups.</p

TrkAIII promotes mitochondrial hydrogen peroxide production.

<p>Line graph displaying comparative levels of H₂0₂ produced over a 210-minute time course by equivalent protein concentrations (60 µg) of intact purified mitochondria from pcDNA, TrkA and TrkAIII SH-SY5Y cells, in an Amplex Red-based assay, following reaction initiation with 10 mM succinate. Results are displayed as the mean±s.d. relative light units produced in minutes following 10 mM succinate addition, in three independent experiments performed in duplicate (asterisks denote statistical significance by t-test comparison of TrkAIII SH-SY5Y cells to either pcDNA or TrkA SH-SY5Y cells).</p