Fig. 3. Combination of IR and CD39 inhibition on GSCs activated DCs by NF-κB p65-NLRP3-IL-1β axis.

A Cells were pretreated with or without 30 μM fludarabine, a STAT1 pathway inhibitor, for 6 h or 100 μM POM-1, a CD39 inhibitor, for 3 h, then irradiated with 10 Gy and cultured for 48 h. Supernatants were collected for ATP measurement. B GSCs were labeled by CFSE, and DCs were labeled by Far Red. GSCs were irradiated with 10 Gy in existence or absence with POM-1 and cultured for 48 h, then co-cultured with DCs for 24 h at a ratio of 1:1 with or without 20 μM A-740003, an ATP receptor inhibitor, pretreatment for 1 h. CD83, a mature DCs marker, on the cell surface was stained and detected using flow cytometry. C Double-positive cells of CFSE and Far Red were DCs that engulfed GSCs. D Supernatants from irradiated GSCs were added into DCs for 24 h, then DCs were collected and NLRP3 level was detected by flow cytometry. E GSCs were pretreated with radiation and POM-1 for 48 h, then co-cultured for 24 h with DCs pretreated with or without 10 μM MCC950, an NLRP3 inhibitor, for 30 min. GSCs were labeled by CFSE, and DCs were labeled by Far red. DCs engulfment was measured using flow cytometry. F DCs were treated with supernatants from pretreated GSCs, and the expressions of phospho-NF-κB p65 and IL-1β were detected using flow cytometry. G Accumulated eATP by CD39 blockade activates NLRP3 inflammasome by P2X7 receptor in DCs. *P < 0.05, **P < 0.01.