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 Ca 2+ influx from the extracellular space or release from intracellular Ca 2+ stores  7) Cell membrane integrity and not metabolic activity should be used for the cell viability assay  8) Unpublished observations should be given in the text or figure  9) Cell viability/membrane integrity following ionomycin and H 2 O 2 stimulation should be provided  10)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? NF-157 (P2RY11 Antagonist) tocris.com 2-methylthioadenosine 5′- monophosphate MeSAMP (P2RY12/13 Antagonist) sigmaaldrich.com

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

11. unpublished observation: –“co-addition of NF-157 or MeSAMP with P2RX7 antagonist A438079 resulted in similar attentuation of BzATP induced VEGF as P2RX7 antagonist alone”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. ADDRESSED

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

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. Primary human monocytes treated for 0.5 or 1 hour with either: Lysates analyzed via RT-PCR 2) P2RX7 inducing VEGF expression? 1.100 μM BzATP 2.300 μM ATP 3.1 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. 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 1.HEPES (control) 2.3 μM antagonist 3.10 μM antagonist 1.HEPES (control) 2.30 μM BzATP 3.100 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. NOT ADDRESSED

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

30. necessary (?)

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

32. NOT ADDRESSED

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

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

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

36. ADDRESSED

37. 5) replicate Figure 1C with ATP?

39. 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 Ca 2+ influx from the extracellular space or release from intracellular Ca 2+ stores

42. ADDRESSED

43. 6) extracellular Ca 2+ influx or intracellular Ca 2+ release? Figure 6C (Revised) Hypothesis: If the P2RX7 stimulation of VEGF release is dependent on the extracellular influx of Ca 2+ mediated through P2RX7, then a cell impermeable Ca 2+ chelator will sequester extracellular Ca 2+ and attenuate or decrease VEGF production

44. 6) extracellular Ca 2+ influx or intracellular Ca 2+ release? ethylene glycol tetraacetic acid (EGTA)

45. 6) extracellular Ca 2+ influx or intracellular Ca 2+ release?

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

47. NOT ADDRESSED

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

49. ADDRESSED

50. 8) unpublished observations ionomycin treatment results

51. 8) unpublished observations H 2 O 2 and combined treatment results:

52. 9) Cell viability/membrane integrity following ionomycin and H 2 O 2 stimulation should be provided

53. NOT ADDRESSED

54. 10) Data in Fig. 1D and Fig. 2 should also be provided as VEGF release in pg/mL

55. NOT ADDRESSED

56. 10) Data in Fig. 1D and Fig. 2 should also be provided as VEGF release in pg/mL NOT ADDRESSED ADDRESSED +

57. 10) addition of data to Fig. 1D and Fig. 2A Figure 2A / Figure 3A (Revised)

58. 10) modification of y-axis in Fig. 1D and Fig. 2A Figure 1D / Figure 2A and B (Revised)

59. 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 Ca 2+ influx from the extracellular space or release from intracellular Ca 2+ 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 H 2 O 2 stimulation should be provided  10) Data in Fig. 1D and Fig. 2 should also be provided as VEGF release in pg/mL

60. 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 Ca 2+ influx from the extracellular space or release from intracellular Ca 2+ 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 H 2 O 2 stimulation should be provided  10) Data in Fig. 1D and Fig. 2 should also be provided as VEGF release in pg/mL

61. 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 Ca 2+ influx from the extracellular space or release from intracellular Ca 2+ 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 H 2 O 2 stimulation should be provided  10) Data in Fig. 1D and Fig. 2 should also be provided as VEGF release in pg/mL

62. Extra Material

63. Propidium iodide Fluorescent molecule Intercalating agent “Leaky” membrane increases fluorescence wikipedia.org