Fig 2: Cell viability of ATP-treated cells in the presence of P2RX inhibitors. TNBC cell lines and MCF-10A cells were treated for 48 hours with increasing concentrations of ATP in the presence of the P2RX inhibitor Iso-PPADS (20 µmol/L), the P2RX7 inhibitor A438079 (20 µmol/L) or the P2RX4 inhibitor 5-BDBD (20 µmol/L) or vehicle addition, and cell viability was measured using the PrestoBlue HS assay. Error bars represent standard deviations calculated from three independent experiments performed in triplicate. One way ANOVA with Tukey’s HSD was applied to ascertain significance. * represents p < 0.05 and ** represents p < 0.01 when comparing vehicle addition to Iso-PPADS, A438079 or 5-BDBD.

Fig 3: Effects of blocking P2X7R on proinflammatory factors in hyperoxia. (a)–(c) Protein levels of TNF-α, IL-1β, and IL-18, determined by ELISA. (d)–(f) Relative mRNA expression of TNF-α, IL-1β, and IL-18, assessed by RT-qPCR. *P < 0.05 and **P < 0.01 versus the control group; # P < 0.05 and ## P < 0.01 versus the hyperoxia group.

Fig 4: Microglia express P2X7R in the Vsp during DED. Male, OVX, and OVXE 14 days DED rats (B,D,F) and sham rats (A,C,E) were perfused with 4% PFA, fixed and brainstem sections stained with anti-Iba1 (red) and dapi (blue). The micrographic examples are of trigeminal subnucleus caudalis (Vc). Scale bar = 80 µm. (G) Densitometry was conducted on Vo, Vi, Vc regions using ImageJ (mean ± SEM). Abbreviations: Ms, male sham; Md, male DED; OvXs, OvX sham; OvXd, OvX DED; OvXEs, OvXE sham; OvXEd, OvXE DED. *p < 0.05 versus sham group. (H) Shaded areas represent regions of trigeminal brainstem that were sampled. N = 4 sections per rat and four rats/group.

Fig 5: Effects of P2X7R/NLRP3 signaling on pyroptosis in HPMVECs. Cells were treated with BzATP (200 µM) to activate P2X7R. Glibenclamide (50 µM) was used to inhibit NLRP3 inflammasome activation. (a) Microscopic images of HPMVECs in control and BzATP treatment groups, with arrows indicating pyroptotic cells. Scale bar = 20 μM. (b)–(e) Protein expression levels of caspase-1 (b), NLRP3 (c), GSDMD (d), and N-GSDMD (e) were determined by WB. GAPDH and β-actin served as loading controls. *P < 0.05 and **P < 0.01 versus the control group; # P < 0.05 and ## P < 0.01 versus the BzATP group.