P2X7RA and B increase ATP release and NFATc1 activity in Te85 transfected cells.

<p>(A): Extracellular ATP was measured in the culture supernatants with ENLITEN rLuciferase/Luciferin reagents as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107224#s2" target="_blank">materials and methods</a>. Te85 clones were plated at 5x10⁵ cells per well in 96 well plates and, following adhesion, incubated for 24 hours in the absence (untreated) or presence of either 100 µM BzATP, 100 µM A740003 or 4 U/ml Apyrase. ATP release was expressed as fold increase over Te85 wt reference sample. Data are means ± SE; N = 3. (B) Nuclear fractions of the different clones were obtained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107224#s2" target="_blank">materials and methods</a>. Activated NFATc1 was measured by ELISA in absence (untreated) or in presence of either 50 µM BzATP or 10 µM cyclosporin (CSA). CSA vehicle (DMSO) was used as control. Results are compared to nuclear NFATc1 levels in Jurkat cells stimulated with PHA supplied by manufacturer. In graph means ± SE of absorbance are shown, N = 9. Colour coding: green Te85 wt, red Te85 P2RX7A, cyan Te85 P2RX7B, purple Te85 P2RX7A+B.</p

Double immunohistochemistry on human osteosarcomas.

<p>Co-immunostainings of osteosarcoma tissue array either with the anti-P2X7R-ec plus anti-Ki67 antibodies (A–D), or with the two primary anti-P2X7R antibodies (E–H) were carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107224#s2" target="_blank">Materials and Methods</a>. (A,C): representative P2X7RB positive osteosarcoma showing a higher number of Ki67 positive nuclei (stained in blue) respect to a representative P2X7RA+B tumour (B,D). (E–H): representative P2X7RA+B positive tumours demonstrating the presence of a mix of cells some (stained in brown) positive for P2X7RB only, some others (stained in blue-brown) positive for both P2X7RA and B. Two different magnifications, 20x (A,B,E,F) and 40x (C,D,G,H) are shown.</p

Schematic summary of obtained data and hypothetical deductions.

<p>Schematic summary of obtained data and hypothetical deductions.</p

Human osteosarcomas express P2X7RA and P2X7RB.

<p>Paraffin embedded osteosarcoma tissue array was assayed by immonohistochemistry for P2X7R expression (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107224#s2" target="_blank">Materials and Methods</a>) and representative samples are shown. (B): osteosarcoma stained with a polyclonal antibody recognizing P2X7R C-terminal domain (anti-P2X7R-Cter). (D): osteosarcoma stained with a monoclonal antibody recognizing P2X7R extracellular domain (anti-P2X7R-ec). (A,C): control samples with appropriate secondary antibodies. (E–H): differential expression of P2X7R isoforms. (E,G): staining with anti-P2X7R-Cter. (F,H): staining with anti-P2XR7-ec. (E,F): representative osteosarcoma stained with both antibodies (P2X7RA+B positive). (G,H): representative osteosarcoma stained only with anti-P2X7Rec (P2X7RB only positive). (I): number of cells per microscopic (40x) field in P2X7RA+B positive (purple) or P2X7RB only positive (cyan) tumour specimens. Cells were counted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107224#s2" target="_blank">Materials and Methods</a>. Data are shown as means ± SE (N = 8, ***p<0.001).</p

P2X7RA and B increase cell growth in Te85 transfected cells.

<p>(A): Te 85 clones were plated at a 10⁵ cells/ml in 6 well plates in cell culture medium without FBS. Percentage of viable cells was evaluated at the different time points as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107224#s2" target="_blank">materials and methods</a>. The graph shows means ± SE, N = 12. *<i>p</i><0.05, ***<i>P</i><0.001 versus Te85 wt control; ^#<i>p</i><0.05 versus Te85 P2RX7A+B, ^$<i>p</i><0.05 versus Te85 P2RX7A. (B) Cell proliferation assessed for 24 hours in absence (untreated) or presence of: 4 U/ml Apyrase, 100 µM A740003, 100 µM BzATP, 100 µM BzATP +10 µM cyclosporin (CSA). A740003 and CSA vehicle (DMSO) was used as control. The graph shows means ± SE, N = 3. Colour coding: green Te85 wt, yellow Te85mock, red Te85 P2RX7A, cyan Te85 P2RX7B, purple Te85 P2RX7A+B.</p

P2X7RA and B expression reduces RANK-L while differently modulates OPG messenger and mineralization.

<p>RANK-L and OPG mRNA levels were evaluated by real-time PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107224#s2" target="_blank">Materials and Methods</a>. Messenger expression was normalized on G3PDH internal control and displayed as fold increase over Te85 wt reference sample. (A): RANK-L mRNA expression (B): OPG mRNA expression. Data are shown as mean ± SE, N = 12, ***p<0.001 versus Te85 wt. (C): Mineralization by Te85 clones was assessed over a 21 days period as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107224#s2" target="_blank">Materials and Methods</a>. (A): Quantification of the amount of alizarin red S staining of each clone. Data are shown as means ± SE (N = 6) analysed by Kruskal-Wallace test with Dunn's multiple comparison post-test. ***<i>P</i><0.001 versus Te85 wt and Te85-P2X7RA; *<i>P</i><0.05 vs Te85 wt; ^$$<i>P</i><0.01 versus Te85 P2X7RA. Colour coding: green Te85 wt, red Te85 P2X7RA, cyan Te85 P2X7RB, purple Te85 P2X7RA+B.</p

Characterization of P2X7R isoforms A and B expressed in Te85 transfected clones.

<p>(A): RT-PCR showing selective expression of P2X7RA or P2X7RB in Te85 clones. Housekeeping control gene was G3PDH, osteoblast specific gene was collagen I. (B): P2X7R surface expression determined by flow cytometry with anti-P2X7R-ec. Graph shows the Mean Fluorescence Intensity (MFI) after subtraction of secondary antibody values. Data are shown as means ± SE, N = 3. ***<i>P</i><0.001 versus Te85 wt; ^###<i>P</i><0.001 versus Te85 P2RX7B; §<i>P</i><0.05 versus Te85 P2RX7A. (C): Representative traces showing intracellular calcium increment following stimulation with 500 µM BzATP. (D): Calcium variations evoked by P2X7R activation showed as means ± SE, N = 10. ***p<0.001 versus Te85 wt. (C): Representative traces showing ethidium bromide uptake following stimulation with 500 µM BzATP. (D): Percentages of ethidium permeabilization on digitonin control showed as means ± SE, N = 10. ***<i>p</i><0.001 versus Te85 wt. Colour coding: green Te85 wt, red Te85 P2X7RA, cyan Te85 P2X7RB, purple Te85 P2X7RA+B.</p