Fig 2: P2X7R isoforms A and B expression in MDS and AML patients.a, b Both P2X7RA and P2X7RB isoforms mRNA are overexpressed in de novo AML as compared to MDS patients. a mRNA was extracted from primary leukemic cells from MDS and de novo AML patients and expression of P2X7R isoform A was compared (MDS, white, n = 10, de novo AML, black, n = 47). b mRNA was extracted from primary leukemic cells from MDS and de novo AML patients and expression of P2X7R isoform B was compared (MDS, white, n = 10, de novo AML, black, n = 47). c, d Within the AML group P2X7R isoforms are differently expressed in relapsing patients as compared to remitting patients. c Expression of P2X7R isoform A within the AML patient’s group by subdividing into de novo (black), relapsing (red), remitting (green) (de novo n = 47, relapsing n = 6, remitting n = 5). d Expression of P2X7R isoform B within the AML patient’s group by subdividing into de novo (black), relapsing (red) and remitting (green). (de novo n = 47, relapsing n = 6, remitting n = 5). Data are represented as mean ± SEM. *P = 0.05; **P = 0.01; ***P = 0.001.

Fig 3: Sk-Mel-28 and Ma-Mel-19 human melanoma cell lines express a functional P2X7 receptor.A mRNA expression of P2X7A and P2X7B in Sk-Mel-28 cells evaluated by real time-PCR (N = 4). B mRNA expression of P2X7A and P2X7B in Ma-Mel-19 cells evaluated by real-time PCR (N = 4). P2X7 receptor activity as a channel was evaluated in C Sk-Mel-28 cells and D Ma-Mel-19 cells by measuring variations in [Ca2+]I following stimulation with P2X7R agonist BzATP (300 µM). The P2X7 receptor antagonists A740003 (20 µM) and AZ10606120 (2 µM) completely obliterated the effect of BzATP. P2X7 activity as a large solute (up to 900 Da) pore was evaluated in E Sk-Mel-28 cells and F Ma-Mel-19 cells by measuring ethidium uptake following stimulation with BzATP (300 µM). The P2X7 receptor antagonists A740003 (20 µM) and AZ10606120 (2 µM) blocked the effect of BzATP.

Fig 4: Immunohistochemistry for P2X7-R-immunoreactivity (IR) in the WT mouse transverse retina is associated with ribbon synapses and colocalises with coventional synapses in the IPL.(A) P2X7-R IR (red) is associated with but does not colocalise with ribbon synapses (ribeye, green) on bipolar cell terminals (VGLUT, blue). (B) Magnified inset from A, arrows indicate ribbon synapses associated with P2X7-R IR puncta. (C) P2X7-R IR (red) colocalises with conventional synapses (bassoon, green) distinct from bipolar cell terminals (VGLUT, blue). (D) Magnified inset from C, arrows indicate conventional synapses that colocalise with P2X7-R IR puncta. Co-localisation analysis indicates that P2X7-R IR puncta do not co-localise with ribbon synapses (E) but do show significant colocalisation with conventional synapses (F) in the IPL. Scale bars are 5 microns for A & C and 2 microns for B & D.

Fig 5: C57bl/6 mice were inoculated into the right flank with B16-F10 cells in wild type and P2X7 null mice. A2AR antagonist SCH58261 (1 mg/kg) or placebo (sterile PBS containing 0.005% DMSO) were intra-peritoneum injected every 3 days after first tumor mass detection (day 5 from inoculum). (A) Ex-vivo tumor volume assessed by calliper. (B) Representative pictures of tumors derived from treated wild type and P2X7 null mice at post inoculum day 14 (n = 7–11 per group). (C–F) A2AR (C) and P2X7R (E) expression by Western Blot and relative quantification (D,F) (n = 5–13 per group). VEGF (G) and TGF-β (H) were evaluated in tumor masses obtained at post inoculum day 14 (n = 3–6 per group). Data are shown as the mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001.