Fig. 7. NTPDase1 deficiency increases P2X₇-associated Yo-Pro-1 uptake by macrophages.

A) ATP dose-dependent Yo-Pro-1 uptake by peritoneal macrophages (Entpd1^−/− [——], Entpd1^+/+ [----]) was evaluated by measuring differences in Yo-Pro-1 mean fluorescence intensity (ΔYo-Pro-1 MFI) using flow cytometry. Cells were incubated with or without ATP (0, 1.0 and 2.0 mM) for the indicated time period. Data show ΔYo-Pro-1 expressed in arbitrary units (AU) for 275–550 cells per 11 seconds. A representative experiment out of 3 is shown. The time point corresponding to ATP addition to the cells (60 sec) is indicated on the graphs by an arrow (↓). Note that statistical analyses were carried out on complete data (n = 15–30 cells per 0.6 sec). Pairwise comparison of individual slope indicate significantly higher intensity growth for Entpd1^−/− when compared to Entpd1^+/+ macrophages (p≤0.002), but slope were significantly different from the baseline slope only when indicated (****, p<0.0001).

B) Effect of KN-62 and apyrase (Apy) on ATP-induced Yo-Pro-1 uptake by peritoneal macrophages (Entpd1^−/− [——], Entpd1^+/+ [----]). The cells were stimulated with 2 mM ATP alone or in combination with KN-62 (3 µM) or Apy (2U) for the indicated period of time. Yo-Pro-1 incorporation measurement and statistical analysis were done as above. Pairwise comparison of individual slope indicate significantly higher intensity growth compared to KN-62 and Apy treatment (****p<0.0001) for both genotype.