Fig. 1.

Extracellular ATP mobilizes Ca²⁺ to activate the NLRP3 inflammasome. (A) LPS-primed BMDMs were loaded with Fura-2 followed by stimulation with 1 mM ATP and analysis of Ca²⁺ flux by time-lapse microscopy. Ca²⁺ is released from intracellular stores during the initial stimulation in Ca²⁺-free buffer, and exchange to 1 mM Ca²⁺-containing buffer permits extracellular Ca²⁺ influx. (B and C) LPS-primed BMDMs were treated or not with Tg for 30 min to deplete ER Ca²⁺ (B) or switched to Ca²⁺ free (−) or Ca²⁺-containing (+) media immediately before ATP stimulation (C). NLRP3 inflammasome activation was assessed by Western blotting of lysates and supernatants (supes) as well as IL-1β ELISA. (D–F) BMDMs were pretreated with XeC, U73122, or 2-APB, followed by ATP stimulation and analysis of Ca²⁺ flux (D) and NLRP3 inflammasome activation (E and F).