Fig 2: Expression of NLRP-1, NLRP-3 and AIM2 in primary hepatic cells. Total RNA from Kupffer cells (KC) cultured for 2 days, freshly isolated sinusoidal endothelial cells (LSEC), portal fibroblasts (pF) in primary culture, hepatic stellate cells (HSC/MFB) cultured for 4 days and hepatocytes (PC) cultured for 2 days were isolated and subjected to quantitative real time PCR for NLRP-1, NLRP-3, and AIM2. In this analysis, the expression of the different genes was set to 100% in KC. The individual primers used for quantitative analysis are depicted in Table 1. For detailed statistical analysis of this set of experiments see Additional file 1.

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: Pyroptosis in primary cortical neurons.a Representative scanning electron microscopy images in sham and oxyHb groups; b Western blot assay for the expression of GSDMD-N in the cell membranes; c Quantification of GSDMD-N; d Representative immunofluorescence staining images of AIM2, GSDMD, caspase-1 and ASC in control and oxyHb-exposed 24 h groups. Scale bars = 50 µm. White arrows show the pores caused by the N-terminal fragment of GSDMD. Bars represent the mean ± S.E.M. ***P < 0.001.

Fig 5: Expression of inflammasome components in rat cirrhotic fat storing cell line CFSC-2G subjected to LPS stimulation. CFSC-2G cells were treated one day after plating with 0, 50, 100 or 200 ng/ml LPS. After 30 min (A), 1 h (B), 2 hrs (C), 4 hrs (D), 8 hrs (E), 16 hrs (F), RNA from respective cells was extracted and, the expression of NLRP-1, NLRP-3. NLRC-4, AIM2, IL-1ß, IL-18, ASC, and TNF-a analysed by qRT-PCR. In this analysis, the expression of each gene without LPS stimulation was set to 1. Please note, the high expression of TNF-a at early (inlets in B and C) and IL-1ß (inlet in D) and medial time points after LPS treatment. For detailed statistical analysis of this set of experiments see Additional file 1.