Fig 2: Membrane blebs induced by P2X7R activation contain TG2. (A) P2X7R signaling induces rapid membrane blebbing. Fluo-4-AM-loaded P2X7R cells were stimulated with BzATP while acquiring fluorescence and phase-contrast images by real-time microscopy to visualize morphological changes and Ca2+ signaling simultaneously (top). Membrane blebs are indicated by arrows. ATP stimulation of parental cells induces oscillating Ca2+ signals but no overt morphological changes (bottom). Scale bar: 25 µm. (B,C) TG2 redistributes into membrane blebs. To confirm export of tagged TG2, TG2- (wild-type, WT) or TG2–GFP-expressing P2X7R cells were stimulated with 100 µM BzATP for 10 min, chased for 30 min in agonist-free medium, followed by analysis of conditioned media and cell extracts for TG2 by western blotting (B). To localize GFP-tagged TG2 during BzATP stimulation, real-time confocal microscopy was employed. Genesis of a membrane bleb is depicted (arrows), with an optical section of GFP fluorescence overlaid onto phase-contrast images to correlate morphological changes with changes in TG2 distribution (C). Scale bar: 10 µm.

Fig 3: Immunohistochemistry for P2X7-R-immunoreactivity (IR) in the WT mouse transverse retina is associated with ribbon synapses and colocalises with coventional synapses in the IPL.(A) P2X7-R IR (red) is associated with but does not colocalise with ribbon synapses (ribeye, green) on bipolar cell terminals (VGLUT, blue). (B) Magnified inset from A, arrows indicate ribbon synapses associated with P2X7-R IR puncta. (C) P2X7-R IR (red) colocalises with conventional synapses (bassoon, green) distinct from bipolar cell terminals (VGLUT, blue). (D) Magnified inset from C, arrows indicate conventional synapses that colocalise with P2X7-R IR puncta. Co-localisation analysis indicates that P2X7-R IR puncta do not co-localise with ribbon synapses (E) but do show significant colocalisation with conventional synapses (F) in the IPL. Scale bars are 5 microns for A & C and 2 microns for B & D.

Fig 4: P2X7-dependent IL-1ß secretion during NADase-deficient Group A Streptococcus infection cannot be explained by altered protein degradation or vesicular release patterns. B6 BMDMs were LPS-primed and infected with wt or nga(G330D) GAS, or left untreated (medium), in the presence of (A) MG-132, (B) 3-MA, or (C) PD150606. (D) Centrifugation setup schematic: supernatants were subjected to sequential centrifugation steps: 300 g, 5min (pre-cfg); 2000 g, 10 min; 10 000 g, 30 min and 100 000 g, 1 h. Pellets were harvested after the 2000 g, 10 000 g and 100 000 g steps. (E) Supernatants and pellets from LPS-primed, infected or untreated (medium) B6 BMDMs were isolated as in (D). IL-1ß levels were measured by ELISA. ATP and Nigericin were used as controls for Nlrp3 inflammasome activation. Graphs show means plus SD for (E) duplicate or (A–C) triplicate technical replicates and are representative of at least three independent experiments. Significant differences are indicated by asterisks as follows: ****P < 0.0001.

Fig 5: P2X7 receptors expression in peritoneal macrophages. (a) Macrophages from uninfected (white bar) and S. mansoni-infected (black bar) mice express similar levels of P2X7 protein. n = 9 replicates from three different animals, for each group. The images above are representative Western blots (U = uninfected and I = infected). (b) P2X7 receptors expression in peritoneal macrophages from control mice (white bar, untreated) or TGF-ß1-treated cells (5 ng/mL, 24 h; black bar) are also similar. The images above are representative Western blots (U = uninfected; TGF-ß1= TGF-ß1-treated macrophages). Lysates from P2X7 KO macrophages were used as negative controls for P2X7 protein expression. Cells lysates were obtained from three animals for each group. (c) Quantitative RT-PCR (qRT-PCR) analysis of P2X7 receptor mRNA levels in peritoneal macrophages. Similar levels of P2X7 mRNA were found in uninfected (white bar), S. mansoni-infected mice (black bar), and TGF-ß1-treated (5 ng/mL, 24 h; hatched bar) mouse macrophages. N = 3–5 samples per group (using different animals).