ATP, ADP and AMP hydrolysis by human MB cell lines.

<p>Confluent cultures of Daoy, ONS76 and D283 cells were incubated with ATP or ADP or AMP as described in the Materials and Methods section. For D283 cells, a concentration of 2.0 mM and an incubation time of 30 minutes were used for all substrates, whereas for Daoy and ONS76 cell lines, 1.0 mM and 30 minutes of incubation to ATP and ADP, and 2.0 mM and 10 minutes of incubation to AMP were used. Specific activities were expressed as nmol Pi/min/mg of protein. Bars represent mean ± SD of four independent experiments performed in triplicate. Data were compared by Two-Way ANOVA test following by Bonferroni post hoc test. (***) p<0.001 and (*) p<0.05 compared to control data.</p

Substrate specificity.

<p>Following confluence Daoy, ONS76 and D283 MB cell lines were incubated with different monophosphonucleosides, as described in Materials and Methods section. Bars represent mean ± SD of 6 independent experiments performed in triplicate. Specific activities were expressed as nmol Pi/min/mg of protein. Data were compared by Two-Way ANOVA test following by Bonferroni post hoc test. (***) p<0.001 and was taken to indicate statistical significance.</p

Basal nucleotide secretion by MB cell lines.

<p>MB cell lines were incubated for 90 minutes with incubation medium in the absence of substrate, as described in Materials and Methods. Extracellular nucleotide/nucleoside concentrations (ATP, ADP, AMP, ADO and INO) were measured by HPLC and concentrations were expressed as µmol product/mg protein (mean±S.D.). Experiments were performed three times in triplicate and the data obtained were analyzed for statistical relevance using a Two-Way ANOVA test followed by the Bonferroni post hoc test. (***) p<0.001 and (*) p<0.05 compared to control data.</p

Flow cytometry analysis of ecto-5′-NT/CD73 protein expression in Daoy, ONS76 and D283 MB cell lines.

<p>Confluent MB cell lines were trypsinized and prepared as described in Materials and Methods. After incubation with purified mouse anti-human CD73 antibody (1∶10) and labeling with Alexa fluor 555 Rabbit anti-mouse IgG (1∶100), cells were analyzed by flow cytometry. (<b>A</b>) Results are shown as the ratio of labeled over non-labeled cells. (<b>B</b>) Representative graphic of ecto-5′NT/CD73-positive cells. Statistical relevance was analyzed by the One-Way ANOVA test following by the Tukey post hoc test. The experiments were performed in triplicate. (**) p<0.001 compared to control data.</p

Analysis of ectonucleotidases expression in MB cell lines.

<p>Total RNA isolated from cell cultures. RT-PCR and real time-PCR reactions were performed as described in Materials and Methods. (<b>A</b>) RT-PCR analysis of E-NPPs, NTPDases, Ecto-5′-NT/CD73, ALP and ADA (<b>B</b>) Quantitative analysis of the relative expression of E-NPPs, NTPDases, Ecto-5′-NT/CD73, ALP and ADA in MB cell lines were performed by real time-PCR where GAPDH expression was used as internal control for normalization of expression levels. Data were analyzed by the One-Way ANOVA test followed by the Tukey post hoc test. The experiments were performed three times in triplicate with (* p<0,01) and (*** p<0.001) indicating statistical relevant difference compared to control data.</p

Ectonucleotidases primer sequences for PCR experiments.

<p>Primer sequences for PCR experiments. These primers listed here were used for both RT-PCR and real time PCR reactions with exception of primers for amplification of NTPDase2 coding sequences. Melting curve analysis was performed to determine the specificity for each real-time PCR reaction.</p

Cytosolic and nuclear P-β-catenin expression in MB cell lines.

<p>Phospho-β-catenin expression was determined by (<b>A</b>) Immunofluorescence and (<b>B</b>) Western blotting assays as described in Materials and Methods. For immunofluorescence, images were captured at objective lens of 20×magnification and the cytosolic and nuclear Phospho-β-catenin immunoreactivity were observed at merge column. For Western blotting, relative Phospho-β-catenin levels of the cytosolic and nuclear fractions obtained by densimetric analysis of protein bands detected in Western blots were compared to GAPDH and HnRNPK expression levels, respectively. Bars represent mean ± SD of 3 independent experiments performed in triplicate. Data were compared by One-Way ANOVA test following by the Tukey post hoc test. (* p<0,01) compared to control data.</p

Extracellular hydrolysis of phosphate esters by MB cell lines.

<p>After the confluence, the MB cells were incubated and the inorganic phosphate amount was analyzed as described in the Materials and Methods. (<b>A</b>) β-glycerophosphate (β-Gli-P), glucose-6-phosphate (Gli-6-P) and inorganic pyrophosphate (PPi), specific substrates to unspecific phosphatases were used to evaluate the enzymatic activity of these enzymes. (<b>B</b>) The MB cells were incubated with AMP in the presence of levamisole, a specific alkaline phosphatase inhibitor, to evaluate the influence of the phosphatases activity in AMP hydrolysis. Specific activities were expressed as nmol Pi/min/mg of protein. Data were compared by Two-Way ANOVA test following by Bonferroni post hoc test. All experiments were performed in triplicate.</p