Fig 2: Effects of inhibited AIM2 inflammasome activation on GSDMD-induced pyroptosis in primary cortical neurons exposed to oxyHb.a, c Western blot assay for the expression of AIM2, GSDMD, GSDMD-N, caspase-1, caspase-1 p20 and ASC in all groups; b, d Quantification of AIM2, GSDMD, GSDMD-N, caspase-1, caspase-1 p20 and ASC; e, f Quantitative analysis of IL-1ß and IL-18 secreted in the supernatant; g Flow cytometry analysis of neurons in all groups; h Quantification of Caspase-1 and PI-positive neurons in all groups. Bars represent the mean ± S.E.M. ***P < 0.001, ****P < 0.0001 and ns means non-significant.

Fig 3: The AIM2 inflammasome-mediated pyroptosis pathway was measured in primary cortical neurons exposed to oxyHb.a Representative images of cultured primary cortical neurons, scale bars (NeuN) = 50 µm, scale bars (MAP2) = 200 µm; b Western blot assay for the expression of AIM2, GSDMD, GSDMD-N, caspase-1, caspase-1 p20 and ASC in all groups; c Quantification of AIM2, GSDMD, GSDMD-N, caspase-1, caspase-1 p20 and ASC; d, e Quantitative analysis of IL-1ß and IL-18 secreted in the supernatant; f Flow cytometry analysis of neurons in all groups; g Quantification of caspase-1 and PI-positive neurons in all groups. Bars represent the mean ± S.E.M. *P < 0.05, ***P < 0.001, ****P < 0.0001 and ns means non-significant. OxyHb oxyhaemoglobin; NeuN neuronal nuclei; MAP2 microtubule associated protein 2; IL-1ß interleukin-1ß; IL-18 interleukin-18; PI propidium iodide.

Fig 4: EPO administration abrogated the activation of the NLRP3, NLRC4, and AIM2 inflammasomes after stroke. A Representative immunoblotting images of NLRP3, NLRC4, AIM2, ASC, CAPS1, IL-1ß and ACTIN after 30 min sham or tMCAo surgery with 6 or 72 h reperfusion B–G Protein-levels of NLRP3, NLRC4, AIM2, ASC, CAPS1, IL-1ß in peri-infarct zone were detected by immunoblotting (n = 4). ACTIN served as loading control. Quantification of protein levels by densitometric analysis. All bars represent means ± SD, individual data points are shown. The time points were blotted separately and normalized to their corresponding NaCl control. For statistical analysis, a 3way ANOVA followed by Tukey’s multiple comparisons test was performed. Comparison of each group at different time-points: #p < 0.05 EPO treatment compared to the NaCl group, *p < 0.05 for intergroup comparison

Fig 5: AIM2 and NLRC4 IHC staining of the late time point of 72 h reperfusion.