Fig 1: FeTPPS attenuated caspase-11-dependent immune responses and lethality in sepsis.A–D WT or Casp11-/- mice were pretreated with FeTPPS 6 mg/kg or saline by intraperitoneal injection 1 h before intraperitoneal challenge with 25 mg/kg LPS. Serum levels of IL-1a, IL-1ß, TNFa, IL-6 (A) (unpaired t-test, n = 5, ****P < 0.0001), western blots for GSDMD-NT, caspase-11 in lung (B), hematoxylin and eosin (H&E) stained sections of lung. Scale bar: 50 µm (C) and kaplan–Meier survival curves (D). Significance assessed using Log-rank test: LPS (WT n = 14) vs. LPS + FeTPPS (WT n = 14), *P = 0.0422; WT LPS (n = 14) vs. Casp11-/- LPS (n = 10), ****P < 0.0001. E–H WT or Casp11-/- mice were pretreated with FeTPPS 6 mg/kg or saline by intraperitoneal injection 1 h before subjected to cecum ligation and puncture (CLP) or sham operation. Serum levels of IL-1a, IL-1ß (E), western blots for GSDMD-NT,Caspase-11 in lung (F), hematoxylin and eosin (H&E) stained sections of lung. Scale bar: 50 µm (G) and kaplan–Meier survival curves (H). Significance assessed using Log-rank test: CLP (WT n = 10) vs. CLP + FeTPPS (WT n = 10), **P = 0.0036; CLP WT (n = 10) vs. Casp11-/- (n = 10), ****P < 0.0001. I WT or Casp11-/- mice were injected with FeTPPS 6 mg/kg 1 h before intraperitoneal challenge with 25 mg/kg LPS. Plasma concentrations of PAI-1 and TAT were measured. An unpaired t-test was used in PAI-1 LPS(WT n = 5) vs. LPS+FeTPPS (WT n = 5), ****P < 0.0001; WT LPS (n = 5) vs. Casp11-/- LPS (n = 5) ***P = 0.0001 and TAT LPS (WT n = 5) vs. LPS + FeTPPS (WT n = 5), *P < 0.0299; WT LPS (n = 5) vs. Casp11-/- LPS (n = 5) ****P < 0.0001.
Fig 2: TSPO interacts with GSDMD during pyroptosis. (A) Multiplex immunohistochemistry assay of TSPO, GSDMD, and CD68 expression in colon tissue from WT and KO mice. Red: TSPO, white: CD68, green: GSDMD; blue, DAPI. White arrows represent for colocalization of TSPO and GSDMD. Scale bars, 20 µm. (B) Left, the subcellular localization of GSDMD in TSPO KO and WT mouse peritoneal macrophages was observed by laser confocal microscopy under an oil microscope. GSDMD was labeled with an anti-GSDMD antibody (red), the cytoskeleton was labeled with phalloidin (green), and the nucleus was labeled with DAPI (blue). Scale bar, 5 µm; right, quantification of GSDMD-p30 positive cells per vision of LPS + ATP activated macrophages. n = 5. (C) The subcellular localization of GSDMD and TSPO in HEK-293T cells was observed by laser confocal microscopy under an oil microscope. GSDMD was labeled with anti-GSDMD antibody (red), TSPO was labeled with GFP (green), and the nucleus was labeled by DAPI (blue). (D) IP verified the interaction between TSPO and GSDMD in pyroptotic macrophages. (E) Microplate assay for detection of H2DCFDA to quantitatively assess reactive oxygen species (ROS) levels in TSPO KO mouse peritoneal macrophages with Ex = 485 nm and Em = 535 nm. (F) Western blot assay to examine the cytochrome C in both cell lysate and supernatant of WT and KO cells. Data expressed as mean ± s.e.m., Student’s t-test, two-sided. * p < 0.05, ** p < 0.01, *** p < 0.001. Three independent experiments were conducted.
Fig 3: VPRH induces cell death via the NLRP3 inflammasome. Approximately 3.5*104 wild-type (B6J), Nlrp1-/-, Nlrp3-/-, and Mlkl-/- BMDMs were seeded into 96-well plates in triplicate, and were primed using LPS (100 ng/ml) for 3 h prior to infection with V. proteolyticus mutants at MOI 20. (A) PI uptake was assessed using real-time microscopy (IncucyteZOOM) and then graphed as the percentage of PI-positive cells normalized to the number of cells in wells. Data are presented as the mean ± SD; n = 3. (B) Summary of normalized AUC for three independent biological experiments shown in panel A. (C-F) Cell lysates and supernatants from experiments described in A were collected 6 h post-infection. (C, D) IL-1ß (C) and TNFa (D) secretion were measured using commercial ELISA kits. (E, F) Inflammasome components and caspase-1 (Casp1), GSDMD, and IL-1ß cleavage were detected in BMDM lysates (E) and supernatants (F) using immunoblots (the numbers on the right of each blot indicate the blot number). The data in A, E, and F are representative of 3 independent experiments. Statistical comparisons in B, C and D between the different bacterial mutants and mouse strains were carried using RM two-way ANOVA, followed by Tukey's multiple comparison test; the results are presented as the mean (bars) ± SD of 3 independent experiments; significant differences are denoted only for comparisons between mouse strains in each infected strain; *P < 0.05, **P < 0.01.
Fig 4: Expression and release of immune mediators in pyroptotic tumor cells. (A) Pattern diagram of immunogenic pyroptosis induced by Tet-On-GSDMD-NT gene expression. (B) The release of LDH (upper) and ATP (lower) was detected during the process of pyroptosis induction. WT+, Vector+, eGFP+ served as a homotype control to eliminate the nonpyroptotic effects of doxy on cells, and G-nondoxy served as a blank control. (C, D) Western blotting showing the expression of GSDMD-NT and (D) the expression and release of HMGB1 was induced in genetically modified TC-1 cells by doxy. (E) PI uptake assays. The modified TC-1 cells were incubated with doxy for 48 h and observed under a fluorescence microscope 6 h after PI dye was added. Scale bar, 50 µm. (F) The release of inflammatory cytokines in the supernatant of pyroptotic tumor cells was detected by ELISA (n=3). (G, H) The expression of HSP70, HSP90 and (H) H-2Kb in the modified TC-1 cells was analyzed by RT-qPCR (n=3). (I, J) Representative flow cytometry pseudocolor dotplots and (J) histogram analysis of H-2Kb expressing cells (n=3) in the dynamic pyroptosis process of the modified TC-1 cells. Graph shows means ± SD; (B, J) One-way ANOVA; (F, G, H) unpaired Student’s t test, *p value < 0.05, **p value < 0.01, ***p value < 0.001, ns represents no significance.
Fig 5: Double KO of Gsdmd and Asc cannot block FlaTox-induced cell death and mouse death.(A to H) Mice of indicated genotypes were intraperitoneally injected with FlaTox (4 µg/g LFn-Fla body weight and 2 µg/g PA body weight) and monitored for survival (A) at indicated times (n = 9). Peritoneal lavages were isolated by using 1 ml PBS at 1 and 3 hours after FlaTox challenge and then subjected to measurements of caspase-1 activity (B), caspase-8 activity (C), caspase-3/7 activity (D), LDH (E), IL-1ß (F), TNF (G), and IL-6 (H) (n = 3). (I to M) Peritoneal macrophages from mice of indicated genotypes were treated with FlaTox (2 µg/ml LFn-Fla and 2 µg/ml PA) for 4 hours. The LDH release (I) and ATP loss (J) were measured. The caspase-1 activity (K), caspase-8 activity (L), and caspase-3/7 activity (M) were measured. Graphs show means ± SE from three independent experiments. (N) Model of the death paths.
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