Implications

1. Implications novel action of P2RX7 signaling aids in monocyte’s role of resolving inflammation via the production of angiogenic factors targeting of P2RX7-dependent VEGF release → tumor treatment (?)

2. Novel action of P2RX7 signaling

3. Tumor treatment Oncogenes induce VEGF release ATP-activated monocytes within tumor environment contribute to tumor angiogenesis Target P2RX7 dependent VEGF release to inhibit tumor growth

4. Reviewer Complaints • 1) VEGF response may also be activated by other P2 receptors • 2) Is P2RX7 only enhancing secretion of VEGF or is inducing VEGF expression? • 3) The kinetics and dose-responses of BzATP-induced VEGF response in the absence and presence of the P2RX7 antagonist should be included • 4) It has been demonstrated before that P2RX7 receptor stimulation leads to VEGF expression by Wei et al. in 2008 • 5) A concentration response curve (dose-response) for ATP should be included for VEGF release • 6) BAPTA-AM use does not differentiate between Ca2+ influx from the extracellular space or release from intracellular Ca2+ stores • 7) Cell membrane integrity and not metabolic activity should be used for the cell viability assay • 8) Unpublished observations should be given in the text or figure • 9) Cell viability/membrane integrity following ionomycin and H2O2 stimulation should be provided • 10) Data in Fig. 1D and Fig. 2 should also be provided as VEGF release in pg/mL

5. 1) The VEGF response may also be activated by other P2 receptors • eg. P2Y11, P2Y13

6. 1) The VEGF response may also be activated by other P2 receptors • eg. P2Y11, P2Y13 ADDRESSED

7. 1) VEGF response activated by other P2 receptors? Figure 3C (Revised) Hypothesis: If the BzATP/ATP stimulation of VEGF production is mediated through P2RY11, then a P2RY11 antagonist will attenuate or decrease VEGF production

8. 1) VEGF response activated by other P2 receptors? Figure 3D (Revised) Hypothesis: If the BzATP/ATP stimulation of VEGF production is mediated through P2RY13, then a P2RY13 antagonist will attenuate or decrease VEGF production

9. 1) VEGF response activated by other P2 receptors? 2-methylthioadenosine 5′-monophosphate MeSAMP (P2RY12/13 Antagonist) NF-157 (P2RY11 Antagonist) tocris.com sigmaaldrich.com

10. 1) VEGF response activated by other P2 receptors?

11. 1) VEGF response activated by other P2 receptors? • unpublished observation: • “co-addition of NF-157 or MeSAMP with P2RX7 antagonist A438079 resulted in similar attentuation of BzATP induced VEGF as P2RX7 antagonist alone”

12. 2) Is P2RX7 only enhancing secretion of VEGF or is inducing VEGF expression?

13. 2) Is P2RX7 only enhancing secretion of VEGF or is inducing VEGF expression? ADDRESSED

14. Quantitative RT-PCR 5’ mRNA Reverse Transcription mRNA 5’ 5’ cDNA 5’ PCR 3’ 5’

15. 2) P2RX7 inducing VEGF expression? Figure 4 (Revised) Hypothesis: If P2RX7 agonists induce VEGF expression, then levels of mRNA encoding VEGF will increase upon treatment with known P2RX7 agonists

16. 2) P2RX7 inducing VEGF expression? • Primary human monocytes treated for 0.5 or 1 hour with either: • Lysates analyzed via RT-PCR 100 μM BzATP 300 μM ATP 1 μg/mL PMA (positive control)

17. 2) P2RX7 inducing VEGF expression? P2RX7 agonists modulate VEGF expression Figure 4A

18. 2) P2RX7 inducing VEGF expression?

19. 2) P2RX7 inducing VEGF expression? • Problems: • Statistical significance tests?

20. 2) P2RX7 inducing VEGF expression? Lingering problem: what if P2RX7 agonists are stimulating other receptors to express VEGF?

21. 2) P2RX7 inducing VEGF expression? Methods (Figure 4B): Pre-incubated monocytes with either: Cells then treated for 1 or 1.5 h with either: VEGF mRNA quantified via RT-PCR HEPES (control) 3 μM antagonist 10 μM antagonist HEPES (control) 30 μM BzATP 100 μM BzATP

22. 2) P2RX7 inducing VEGF expression? Figure 4B

23. 2) P2RX7 inducing VEGF expression? Figure Caption:

24. 2) P2RX7 inducing VEGF expression? Results:

25. 2) P2RX7 inducing VEGF expression? Figure 4B: Does not match the results section Does not match the figure caption

26. 2) P2RX7 inducing VEGF expression? Results (Figure 4B): Agonist induced VEGF expression is dependent upon P2RX7 activation

27. 3) The kinetics and dose-responses of BzATP-induced VEGF response in the absence and presence of the P2RX7 antagonist should be included

28. 3) The kinetics and dose-responses of BzATP-induced VEGF response in the absence and presence of the P2RX7 antagonist should be included NOT ADDRESSED

29. 3) replicate Figure 1B and 1C with P2RX7 antagonist?

30. 3) replicate Figure 1B and 1C with P2RX7 antagonist? • necessary (?)

31. 4) It has been demonstrated before that P2RX7 receptor stimulation leads to VEGF expression by Wei et al. in 2008

32. 4) It has been demonstrated before that P2RX7 receptor stimulation leads to VEGF expression by Wei et al. in 2008 NOT ADDRESSED

33. 4) past research already demonstrated P2RX7 relationship w/ VEGF? Original Hill Paper Revised Hill Paper

34. 4) past research already demonstrated P2RX7 relationship w/ VEGF? Original Hill Paper Revised Hill Paper

35. 5) A concentration response curve (dose-response) for ATP should be included for VEGF release

36. 5) A concentration response curve (dose-response) for ATP should be included for VEGF release ADDRESSED

37. 5) replicate Figure 1C with ATP?

38. 5) replicate Figure 1C with ATP?

39. 5) replicate Figure 1C with ATP? VEGF release from each patient after 100 μM BzATP

40. 5) replicate Figure 1C with ATP? • problems with revised Figure 1D: • no statistical tests performed

41. 6) BAPTA-AM use does not differentiate between Ca2+ influx from the extracellular space or release from intracellular Ca2+ stores

42. 6) BAPTA-AM use does not differentiate between Ca2+ influx from the extracellular space or release from intracellular Ca2+ stores ADDRESSED

43. 6) extracellular Ca2+ influx or intracellular Ca2+ release? Figure 6C (Revised) Hypothesis: If the P2RX7 stimulation of VEGF release is dependent on the extracellular influx of Ca2+ mediated through P2RX7, then a cell impermeable Ca2+ chelator will sequester extracellular Ca2+ and attenuate or decrease VEGF production

44. 6) extracellular Ca2+ influx or intracellular Ca2+ release? ethylene glycol tetraacetic acid (EGTA)

45. 6) extracellular Ca2+ influx or intracellular Ca2+ release?

46. 7) Cell membrane integrity and not metabolic activity should be used for the cell viability assay

47. 7) Cell membrane integrity and not metabolic activity should be used for the cell viability assay NOT ADDRESSED

48. 8) Unpublished observations should be given in the text or figure

49. 8) Unpublished observations should be given in the text or figure ADDRESSED

50. 8) unpublished observations • ionomycin treatment results