Fig. 7: Intracellular calcium signaling through P2YR is required for NLRP3 inflammasome activation.

a Video-microscopy recording of intracellular calcium during hypotonic challenge in control and LRRC8A-KD THP-1 cells expressing GCaMP6-P2A-mScarlet, with or without P2Y2 antagonist ARC118925 (10 µM). b Quantification from (a) showing area under the peaks averaged and normalized to control. Data are mean ± SEM from n = 5 experiments for control and LRRC8A-KD, n = 2 experiments for ARC118925. 30–60 cells per experiment. c Chloride currents in control THP-1 cells in isotonic (300 mOsm.l−1) and hypotonic (220 mOsm.l-1) NMDGCl solutions, with/without ARC118925 (10 µM). I/V curves show mean current density under hypotonic conditions with ARC118925, MRS2578, or MCC950 (10 µM). Pipette solution contained NMDGCl. Data are mean ± SEM from 4 to 15 cells, n = 4–15 experiments. d–f Simultaneous intracellular calcium signals and cell surface analysis during hypotonic challenge at single-cell level. d Combined recording of cell surface variation (mScarlet) and intracellular calcium (GCaMP6). Scale bar 20 µm. e Single-cell analysis shows RVD initiation coincides with a burst of intracellular calcium release. f Time correlation between RVD initiation and peak of intracellular calcium signal. d–f: n = 2 experiments, analysis of 70 cells. g Left panel: cartoon of PLC pathway activation by Gq-coupled P2YR leading to intracellular calcium mobilization. Middle and right panels: ELISA results of IL-1β levels in supernatants of control THP-1 cells exposed to MSU or CPP crystals (200 μg/ml, 4 h) with/without PLC inhibitor (U73122, 5 μM) or its inactive analog (U73343, 5 μM). n = 3 independent experiments. Values are mean ± SEM. Two Way ANOVA with Tukey’s multiple comparison post hoc test. Panel g Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en.