Fig 1: Analysis of non-canonical inflammasome pathway.A LDH cytotoxicity assay in the presence of LPS or SCGB3A2 alone, or the two together (SCGB3A2 + LPS). B LDH cytotoxicity assay in the presence of various amount of nigericin. Average ± SD from more than three independent experiments. *P < 0.01, **P < 0.0001 by Tukey’s multiple comparison. C Morphology of cells cultured in the presence of LPS or SCGB3A2 alone, SCGB3A2 + LPS, or nigericin for 3 h. For nigericin-treated group, cells were primed with LPS before addition of nigericin. Black arrow indicates ballooned cells, characteristic feature of pyroptosis. D Western blotting for the cleaved forms of CASP1 (p20), CASP4 (p20), and GSDMD (N-terminal). S: SCGB3A2, N: nigericin. Experiments were repeated more than twice and same results were obtained.
Fig 2: ROS production leads to NLRP3 inflammasome activation-mediated endothelial pyroptosis and apoptosis. Mouse pulmonary endothelial cells were exposed to LPS (1 µg/ml) in the presence or absence of the ROS scavenger NAC (5 mM) for 6 h. (A) Representative protein bands of NLRP3, casp-1(p20) and GSDMD-N. Casp-1(p20) was normalized to pro-casp-1, while GSDMD-N was normalized to GSDMD-FL. (B) Representative western blot bands of Bax and Bcl-2. (C) Representative TUNEL-positive cells (red); nuclei were counterstained with DAPI (blue). Scale bar, 100 µm. Data are expressed as the mean ± standard error of the mean (n=4). *P<0.05, **P<0.01 vs. con; ##P<0.01 vs. LPS. ROS, reactive oxygen species; NLRP3, NOD-, LRR- and pyrin domain-containing protein 3; LPS, lipopolysaccharide; GSDMD-N, gasdermin D N-terminal fragment; NAC, N-acetyl-L-cysteine; FL, full length; Con, control; Cit, citrulline; ROS, reactive oxygen species; casp, caspase.
Fig 3: Cit protects against LPS-induced pulmonary pyroptosis and apoptosis. (A) IL-1ß and (B) IL-18 concentration in lung tissue was determined using ELISA kits. (C) Expression of GSDMD-N was normalized to GSDMD-FL. (D) Representative western blot bands of Bax and Bcl-2. (E) Representative TUNEL-positive cells (green) in lung sections. Cell nucleus was stained with DAPI (blue). Scale bar, 100 µm. Data are expressed as the mean ± standard error of the mean (n=6). **P<0.01 vs. con; ##P<0.01 vs. LPS. LPS, lipopolysaccharide; GSDMD, gasdermin D; N, N-terminal; FL, full length; Con, control; Cit, citrulline.
Fig 4: Schematic model: PRRSV infection induces NLRP3 inflammasome activation and pyroptosis. Signal I (priming; left side) is provided by PRRSV infection, leading to the transcriptional upregulation of NLRP3 inflammasome components and pro-IL-1ß. Signal II (activation; right side) is provided by the PRRSV RTC-induced dispersed trans-Golgi network (dTGN), which activates NLRP3, followed by the recruitment of ASC and CASP1 to form the NLRP3 inflammasome complex. Formation of the NLRP3 inflammasome activates CASP1, which in turn cleaves pro-IL-1ß and GSDMD. After cleavage, the N terminus of GSDMD (GSDMD-NT) inserts into the membrane, forming pores and inducing pyroptosis.
Fig 5: PRRSV infection causes PAM pyroptosis in vivo. (A) Representative micrographs of lungs obtained from mock- and PRRSV-infected piglets (7, 14, and 21 days postinfection [dpi]) showing histopathological changes. Lung sections were stained with hematoxylin and eosin (H&E). Solid and thin arrows indicate hemorrhage and hyperemia or infiltration of inflammatory cells within alveolar spaces and alveolar septum. Triangle indicates interlobular septal thickening, inflammatory cell infiltration around the bronchiole, or narrowed alveolar spaces. Solid triangle indicates inflammatory cells, necrotic debris, and exfoliated epithelial cell infiltrate in the alveolar and bronchiole. No significant changes in the lungs of mock-infected pigs. (B) Immunohistochemistry (IHC) was used to examine the PRRSV-N antigen in piglet lung tissues, using anti-PRRSV-N antibody. Arrowhead indicates PRRSV-N antigen signals in macrophages located within or around alveolus and bronchus. (C and D) Scores of microscopic lung lesions and PRRSV-N antigen for piglets infected with PRRSV. (E) Immunohistochemistry was used to examine the activated capsase-1 (CASP1) in piglet lung tissues, using anti-cleaved-CASP1 antibody. Red arrow indicates cleaved-CASP1 signals. (F and G) Enzyme-linked immunosorbent assay (ELISA) was used to measure IL-1ß levels in serum and bronchoalveolar lavage fluid (BALF) from mock- and PRRSV-infected piglets. (H) Immunoblotting analysis was used to assess GSDMD cleavage in lung tissues of piglets with and without PRRSV infection. GSDMD-N is the N-terminal cleavage product of GSDMD. Significant differences from mock-infected piglets are shown as ***, P < 0.001 (one-way ANOVA). Data are representative of three experiments (mean ± SEM of n = 6 or n = 3).
Supplier Page from Abcam for Anti-GSDMD antibody