Journal: PLoS Pathogens

Article Title: The colonic pathogen Entamoeba histolytica activates caspase-4/1 that cleaves the pore-forming protein gasdermin D to regulate IL-1β secretion

doi: 10.1371/journal.ppat.1010415

Figure Lengend Snippet: ( A, C, E ) WT, CRISPR/Cas9 CASP1 KO , and CRISPR/Cas9 CASP4 KO macrophages were incubated for increasing amounts of time with 1:20 Eh to macrophage ratio. LPS (50 ng/mL) and NGC (10 μM) for 60 min acted as a positive control. Cells were washed, lysed and equal amount of lysates (LYS) was loaded onto SDS-PAGE and immunoblot analysis was performed for GSDMD cleavage that present in cell lysates. ( B, D, F ) Quantifications of GSDMD p30 protein were performed by densitometric analysis and blots were reprobed for GAPDH. Negative cells only acted as an internal control. Data and immunoblots are representative of at least three independent experiments (n = 3) and statistical significance was calculated using with an ANOVA and Bonferroni’s post-hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Bars represent mean ± SEM.

Article Snippet: Human recombinant GSDMD was obtained from Origene, and recombinant caspase-1 and recombinant caspase-4 were purchased from Enzo life sciences.

Techniques: CRISPR, Incubation, Positive Control, SDS Page, Western Blot, Control

Journal: PLoS Pathogens

Article Title: The colonic pathogen Entamoeba histolytica activates caspase-4/1 that cleaves the pore-forming protein gasdermin D to regulate IL-1β secretion

doi: 10.1371/journal.ppat.1010415

Figure Lengend Snippet: ( A ) WT, CRISPR/Cas9 CASP1 KO and CRISPR/Cas9 CASP4 KO macrophages were incubated with Eh for 30 min or the positive control LPS (50 ng/mL) and NGC (10 μM) for 60 min. Cell supernatant (SN) was TCA precipitated and cells were washed and lysed. Equal amount of supernatants and lysates (LYS) was loaded onto SDS-PAGE and immunoblot analysis was performed for caspase-4 and caspase-1 secreted to the supernatants and along with GSDMD p30 pore-forming fragment in lysates, and blots were reprobed for GAPDH. ( B ) Quantifications of cleaved GSDMD were performed by densitometric analysis from three independent experiments, and the negative (cells only) acted as an internal control. ( C ) Cell supernatant was added to HEK-Blue reporter cells to detect bioactive IL-1β by measuring SEAP. ( D ) THP-1 cell supernatant was assessed by the release of LDH following Eh stimulation and normalized to non-stimulated negative controls (basal cell death). Data and immunoblots are representative of at least three independent experiments (n = 3) and statistical significance was calculated with ANOVA and Bonferroni’s post-hoc test between WT and KO macrophages and between CASP1KO and CASP4 KO macrophages at each time point. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Bars represent mean ± SEM.

Article Snippet: Human recombinant GSDMD was obtained from Origene, and recombinant caspase-1 and recombinant caspase-4 were purchased from Enzo life sciences.

Techniques: CRISPR, Incubation, Positive Control, SDS Page, Western Blot, Control

Journal: PLoS Pathogens

Article Title: The colonic pathogen Entamoeba histolytica activates caspase-4/1 that cleaves the pore-forming protein gasdermin D to regulate IL-1β secretion

doi: 10.1371/journal.ppat.1010415

Figure Lengend Snippet: ( A, C ) ASC def and CRISPR/Cas9 NLRP3 KO macrophages were incubated with Eh from 5 min to 60 min or the positive control, LPS (50 ng/mL) and NGC (10 μM) for 60 min. Equal amount of cell lysates (LYS) was loaded onto SDS-PAGE and immunoblot analysis was performed for GSDMD p30 pore-forming fragment presented in lysates and blots were reprobed with GAPDH. ( B, D ) Quantification of cleaved GSDMD p30 fragment was performed by densitometric analysis from three independent experiments, and the negative (cells only) acted as an internal control. ( E ) Bioactive IL-1β levels were quantified by SEAP assay that detected in HEK-Blue reporter cells. ( F ) Cell death was quantified by LDH release into the culture supernatant and is shown as a percentage of LDH release compared to non-stimulated cells (control). ( E, F ) Statistical significance was calculated between WT and ASC def, WT and NLRP3 KO as well as between ASC def and NLRP3 KO macrophages at each time point. Data and immunoblots are representative of at least three independent experiments (n = 3) and statistical significance was calculated with ANOVA and Bonferroni’s post-hoc test (* p < 0.05, ** p < 0.01, **** p < 0.0001). Bars represent mean ± SEM.

Article Snippet: Human recombinant GSDMD was obtained from Origene, and recombinant caspase-1 and recombinant caspase-4 were purchased from Enzo life sciences.

Techniques: CRISPR, Incubation, Positive Control, SDS Page, Western Blot, Control, SEAP Assay

Journal: PLoS Pathogens

Article Title: The colonic pathogen Entamoeba histolytica activates caspase-4/1 that cleaves the pore-forming protein gasdermin D to regulate IL-1β secretion

doi: 10.1371/journal.ppat.1010415

Figure Lengend Snippet: ( A ) WT and CRISPR/Cas9 GSDMD KO macrophages were incubated with Eh for 10 and 30 min, respectively. LPS (50 ng/mL) and NGC (10 μM) stimulation for 60 min acted as the positive control. Cell supernatant (SN) was TCA precipitated and cells were washed and lysed. Equal amount of supernatants and lysates (LYS) was resolved on SDS-PAGE and immunoblot analysis was performed for GSDMD, caspase-4 and caspase-1 in both the cell lysates and supernatants, and blots were reprobed for GAPDH. ( B ) Quantifications of active caspase-4 proteins were performed by densitometric analysis from three independent experiments, and the negative (cells only) acted as an internal control. ( C ) Cell supernatant from stimulated macrophages was added to HEK-Blue reporter cells to detect bioactive IL-1β via the SEAP assay in both WT and CRISPR/ Cas9 GSDMD KO macrophages that were incubated with Eh for increasing amounts of time. ( D ) Cell death was determined by LDH assay using supernatant from stimulated macrophages, and is shown as a percentage of LDH release compared to non-stimulated cells (control). Data and immunoblots are representative of three separate experiments (n = 3) and a one-way ANOVA was used to determine statistical significance of differences between WT and GSDMD KO cells at each treatment time (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Bars represent mean ± SEM.

Article Snippet: Human recombinant GSDMD was obtained from Origene, and recombinant caspase-1 and recombinant caspase-4 were purchased from Enzo life sciences.

Techniques: CRISPR, Incubation, Positive Control, SDS Page, Western Blot, Control, SEAP Assay, Lactate Dehydrogenase Assay

Journal: PLoS Pathogens

Article Title: The colonic pathogen Entamoeba histolytica activates caspase-4/1 that cleaves the pore-forming protein gasdermin D to regulate IL-1β secretion

doi: 10.1371/journal.ppat.1010415

Figure Lengend Snippet: ( A ) Necrosulfonamide (NSA) as a direct chemical inhibitor of GSDMD, binds directly to GSDMD, inhibiting oligomerization of GSDMD p30 pore-forming fragment to inhibit pyroptosis. ( B ) NSA was added to macrophages 60 min prior to Eh stimulation. Since NSA was prepared in dimethyl sulfoxide (DMSO), DMSO, NSA only (20 μM) were used to detect if DMSO or NSA itself would have any effect on THP-1 cells, and unstimulated cells acted as the negative control. After Eh stimulation, cell free supernatant was added to HEK-Blue IL-1β reporter cells to detect bioactive IL-1β using the SEAP assay. ( C ) Pyroptotic pore formation and cell death were assessed through LDH release, cell free supernatants from the same experiments were used to quantify LDH released into the culture supernatant and is shown as a percentage of LDH release compared to non-stimulated cells. ( B, C ) Eh treatment only as a positive control and statistical significance was calculated between Eh 30 min and various concentration of NSA treatments. ( D ) Cell free supernatant was added to HEK-Blue IL-1β reporter cells to detect bioactive IL-1β using the SEAP assay to detect NSA inhibition in GSDMD pore formation in WT, CASP1 KO , CASP4 KO macrophage. LPS (50 ng/mL) and NGC (10 μM) stimulation for 60 min acted as the positive control. Statistical significance was calculated between WT and KO macrophages and between CASP1KO and CASP4 KO macrophages at each time point. ( E-G ) Immunoblot analysis was performed for GSDMD p30 cleavage in cell lysates (LYS), and blots were reprobed for GAPDH. WT, CRISPR/Cas9 CASP1 KO and CRISPR/Cas9 CASP4 KO macrophages were pre-incubated with NSA for 60 min before stimulation with LPS + NGC. Data and immunoblots are representative of six experiments (n = 6) and statistical significance was calculated with Student’s t-test and one-way ANOVA followed by post hoc Bonferroni test, (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Bars represent mean ± SEM.

Article Snippet: Human recombinant GSDMD was obtained from Origene, and recombinant caspase-1 and recombinant caspase-4 were purchased from Enzo life sciences.

Techniques: Negative Control, SEAP Assay, Positive Control, Concentration Assay, Inhibition, Western Blot, CRISPR, Incubation

Journal: PLoS Pathogens

Article Title: The colonic pathogen Entamoeba histolytica activates caspase-4/1 that cleaves the pore-forming protein gasdermin D to regulate IL-1β secretion

doi: 10.1371/journal.ppat.1010415

Figure Lengend Snippet: ( A ) GST and His-tagged recombinant GSDMD (rGSDMD) was incubated with 1U recombinant caspase-1 (rC-1) and recombinant caspase-4 (rC-4) at 37°C with indicated time points, following the detection by immunoblot analysis. ( B ) rGSDMD were incubated for 15 min at 37°C with active rC-1 and rC-4 in absence or presence of inhibitor Z-VAD-fmk and Z-YVAD-fmk (100 μM, 10 min, room temperature) and rGSDMD cleavage was assessed by western blot with anti-GSDMD, anti-GST and anti-His antibody. ( C ) Macrophages were immunoprecipitated with anti-GSDMD antibody and immunoprecipitants were incubated with active rC-1 and rC-4 for 16 h at 37°C and GSDMD cleavage was assessed by western blotting identified with anti-GSDMD antibody. Direct cell lysate was used as a control (Ctrl). ( D ) C terminal Myc-DDk-tagged human GSDMD plasmid was overexpressed in HEK 293T cells and, ( E ) Basal expression of GSDMD was detected via anti-GSDMD antibody. ( F-H ) HEK 293T cells transfected with Myc-DDk-tagged GSDMD plasmid were immunoprecipitated with anti-DYKDDDDK tag antibody. Immunoprecipitants were incubated with active rC-4 and rC-1 for 16 h at 37°C and GSDMD cleavage was assessed by western blot with anti-DYKDDDDK tag and anti-GSDMD antibody. Immunoblots are representative of at least three independent experiments (n = 3).

Article Snippet: Human recombinant GSDMD was obtained from Origene, and recombinant caspase-1 and recombinant caspase-4 were purchased from Enzo life sciences.

Techniques: Recombinant, Incubation, Western Blot, Immunoprecipitation, Control, Plasmid Preparation, Expressing, Transfection

Journal: PLoS Pathogens

Article Title: The colonic pathogen Entamoeba histolytica activates caspase-4/1 that cleaves the pore-forming protein gasdermin D to regulate IL-1β secretion

doi: 10.1371/journal.ppat.1010415

Figure Lengend Snippet: The molecular weight of full length rGSDMD is 81 kDa as a GST tag is linked to NT of rGSDMD, whereas CT is tagged by His. ( A ) Cleavage of 5 μg purified recombinant GSDMD with 6U recombinant caspase-4 visualized by Coomassie blue staining of the protein bands that were excised (white dotted box) and sequenced by Edman degradation. Immunoblot is representative of at least three independent experiments (n = 3). ( B ) Schematic representation showed full length rGSDMD with tags. Red line indicated GST tag attached to the NT, while CT was linked by a His tag in grey, and full GSDMD sequence is marked in black. ( C ) Caspase-4 cleavage sequence logo was generated from “MEROPS” ( https://www.ebi.ac.uk/merops/ ) based on the peptidase database. ( D ) After alignment of the calls that we obtained from Edman degradation, the predicated cleavage for caspase-4 on GSDMD marked the same cleavage site as caspase-1 (black arrow). ( E ) NT GST-tagged rGSDMD incubated with active rC-4 for 16 h at 37°C and degraded fragments generated from full length GSDMD were evaluated by immunoblot analysis followed by Coomassie blue staining. Edman degradation analysis of the 26 kDa band after alignment of amino acid calls suggested a cleavage site for caspase-4 on GSDMD at aspartic acid 275 (D275) position.

Article Snippet: Human recombinant GSDMD was obtained from Origene, and recombinant caspase-1 and recombinant caspase-4 were purchased from Enzo life sciences.

Techniques: Molecular Weight, Purification, Recombinant, Staining, Western Blot, Sequencing, Generated, Incubation

Journal: PLoS Pathogens

Article Title: The colonic pathogen Entamoeba histolytica activates caspase-4/1 that cleaves the pore-forming protein gasdermin D to regulate IL-1β secretion

doi: 10.1371/journal.ppat.1010415

Figure Lengend Snippet: ( A ) Preparation and workflow for proteomic analysis. ( B ) Metascape analysis of different pathways within control and Eh -contacted hyperactivated macrophages. Some upregulated pathways in red and downregulated pathways in blue are what we considered most relevant and interesting. ( C ) Some interesting proteins involved in downregulated and upregulated pathways were characterized, and the common proteins were also indicated. ( D ) Macrophages were incubated with Eh (20:1) for 10 and 30 min to detect NINJ1 protein level and blots were reprobed for GAPDH. Immunoblots are representative of at least three independent experiments (n = 3). ( E ) STRING analysis of GSDMD protein-protein interaction, and NINJ1 protein-protein interaction with other top hits proteins were conducted.

Article Snippet: Human recombinant GSDMD was obtained from Origene, and recombinant caspase-1 and recombinant caspase-4 were purchased from Enzo life sciences.

Techniques: Control, Incubation, Western Blot

Journal: PLoS Pathogens

Article Title: The colonic pathogen Entamoeba histolytica activates caspase-4/1 that cleaves the pore-forming protein gasdermin D to regulate IL-1β secretion

doi: 10.1371/journal.ppat.1010415

Figure Lengend Snippet: The activation of caspase is initially triggered by Eh in contact with macrophage via the Gal-lectin to Gal/GalNAc residues on the surface of macrophage. Eh CP-A5 is highly expressed on the surface of Eh and following Gal-lectin binding brings, Eh CP-A5 RGD sequences ligate α 5 β 1 integrin on the macrophage surface to induce the generation of ATP and release through the opening of pannexin-1 channel that subsequently signals back onto the P2X 7 receptor to activate the NLRP3 inflammasome. Simultaneously, K + efflux and the production of ROS collaborate to activate the NLRP3 inflammasome. The NLRP3 inflammasome in turn activates caspase-1, whereas, the activation of caspase-4 is independent of the inflammasome complex. Whereas both caspase-4/1 acted together to induce the cleavage of GSDMD, caspase-4 played a dominant role in this process. The cleaved GSDMD initiates pore formation allowing bioactive IL-1β release without causing significant cell pyroptosis.

Article Snippet: Human recombinant GSDMD was obtained from Origene, and recombinant caspase-1 and recombinant caspase-4 were purchased from Enzo life sciences.

Techniques: Activation Assay, Binding Assay

Journal: Cell death discovery

Article Title: EZH2-STAT3 signaling pathway regulates GSDMD-mediated pyroptosis in glioblastoma.

doi: 10.1038/s41420-024-02105-0

Figure Lengend Snippet: Fig. 2 Correlation Among EZH2, STAT3, cell cycle, and pyroptosis. A Biological processes (BP), B molecular functions (MF), and C cellular components (CC) are mostly related to EZH2 in the TCGA database. D Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of EZH2 in the TCGA database. E Correlation analysis of EZH2 and apoptosis gene set, STAT3 and apoptosis gene set, apoptosis gene set, and cell cycle gene set. F Kaplan–Meier curves for cell cycle and apoptosis in GBM + LGG. G Protein–protein interaction (PPI) network analysis for EZH2, STAT3, and GSDMD.

Article Snippet: Antibodies were sourced from the following providers: Cell Signaling Technology (Danvers, Massachusetts, USA)—anti-EZH2 (1:1000), anti- STAT3 (1:1000), anti-phospho (p)-STAT3 (1:1000); Affinity Biologicals (Jiangsu, China)—anti-NLRP3 (1:1000); Abcam (Cambridge, MA, USA)— anti-GSDMD (1:1000), anti-cleaved N-terminal GSDMD (1:1000); Proteintech (Wuhan, China)—Beta Actin Recombinant antibody (1:20,000), IL-1 Beta Polyclonal antibody, IL-18 Polyclonal antibody; secondary antibodies (goat anti-rabbit and goat anti-mouse).

Techniques:

Journal: Cell death discovery

Article Title: EZH2-STAT3 signaling pathway regulates GSDMD-mediated pyroptosis in glioblastoma.

doi: 10.1038/s41420-024-02105-0

Figure Lengend Snippet: Fig. 3 EZH2, as an upstream regulator of STAT3, regulates pyroptosis through the inflammasome. A Fluorescence microscopy observes the localization of EZH2 (green) and p-STAT3 (red) in U87. B, C RT-qPCR detects the expression levels of EZH2 and STAT3 in HA1800, U87, H4, and A172. D, E Protein blotting shows the protein levels of STAT3, p-STAT3, and NLRP3 in U87 and LN229 after DZNep treatment. F, G Protein blotting reveals increased protein levels of NLRP3, GSDMD, and N-GSDMD in U87 and LN229 after DZNep treatment. For D–G, data are presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, N.S. not significant by t-test.

Article Snippet: Antibodies were sourced from the following providers: Cell Signaling Technology (Danvers, Massachusetts, USA)—anti-EZH2 (1:1000), anti- STAT3 (1:1000), anti-phospho (p)-STAT3 (1:1000); Affinity Biologicals (Jiangsu, China)—anti-NLRP3 (1:1000); Abcam (Cambridge, MA, USA)— anti-GSDMD (1:1000), anti-cleaved N-terminal GSDMD (1:1000); Proteintech (Wuhan, China)—Beta Actin Recombinant antibody (1:20,000), IL-1 Beta Polyclonal antibody, IL-18 Polyclonal antibody; secondary antibodies (goat anti-rabbit and goat anti-mouse).

Techniques: Fluorescence, Microscopy, Quantitative RT-PCR, Expressing

Journal: Cell death discovery

Article Title: EZH2-STAT3 signaling pathway regulates GSDMD-mediated pyroptosis in glioblastoma.

doi: 10.1038/s41420-024-02105-0

Figure Lengend Snippet: Fig. 4 Inhibition of STAT3 activates pyroptosis and suppresses glioma cell proliferation. A, B Detection of STAT3 and p-STAT3 expression levels in cells after 48 h treatment with SH-4–54 (10 μM). C, D Protein blotting shows the protein levels of NLRP3, GSDMD, and N-GSDMD in U87 and LN229 after SH-4–54 treatment. E Morphological changes in U87 cells after SH-4–54 treatment. F Increased lactate dehydrogenase (LDH) levels in the culture medium after 48 h SH-4–54 treatment. G, H EdU assay demonstrates decreased proliferation of U87 and LN229 cells after SH-4–54 treatment. For A–D, data are presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, N.S. not significant by t- test.

Article Snippet: Antibodies were sourced from the following providers: Cell Signaling Technology (Danvers, Massachusetts, USA)—anti-EZH2 (1:1000), anti- STAT3 (1:1000), anti-phospho (p)-STAT3 (1:1000); Affinity Biologicals (Jiangsu, China)—anti-NLRP3 (1:1000); Abcam (Cambridge, MA, USA)— anti-GSDMD (1:1000), anti-cleaved N-terminal GSDMD (1:1000); Proteintech (Wuhan, China)—Beta Actin Recombinant antibody (1:20,000), IL-1 Beta Polyclonal antibody, IL-18 Polyclonal antibody; secondary antibodies (goat anti-rabbit and goat anti-mouse).

Techniques: Inhibition, Expressing, EdU Assay

Journal: Cell death discovery

Article Title: EZH2-STAT3 signaling pathway regulates GSDMD-mediated pyroptosis in glioblastoma.

doi: 10.1038/s41420-024-02105-0

Figure Lengend Snippet: Fig. 5 EZH2–STAT3 activation of pyroptosis accompanied by inflammatory factor production. A, B RT-qPCR detects IL-1β and IL-18 expression levels in U87 and LN229 cells after DZNep treatment. C–F Protein blotting shows IL-1β and IL-18 protein levels in U87 and LN229 cells after DZNep and SH-4–54 treatment. G Heatmap displaying GSDMD expression and immune function enrichment scores in TCGA databases. R‐value and P‐value of the correlation analysis are shown on the right. For C–F, data are presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, N.S. not significant by t-test.

Article Snippet: Antibodies were sourced from the following providers: Cell Signaling Technology (Danvers, Massachusetts, USA)—anti-EZH2 (1:1000), anti- STAT3 (1:1000), anti-phospho (p)-STAT3 (1:1000); Affinity Biologicals (Jiangsu, China)—anti-NLRP3 (1:1000); Abcam (Cambridge, MA, USA)— anti-GSDMD (1:1000), anti-cleaved N-terminal GSDMD (1:1000); Proteintech (Wuhan, China)—Beta Actin Recombinant antibody (1:20,000), IL-1 Beta Polyclonal antibody, IL-18 Polyclonal antibody; secondary antibodies (goat anti-rabbit and goat anti-mouse).

Techniques: Activation Assay, Quantitative RT-PCR, Expressing

Journal: Cell death discovery

Article Title: EZH2-STAT3 signaling pathway regulates GSDMD-mediated pyroptosis in glioblastoma.

doi: 10.1038/s41420-024-02105-0

Figure Lengend Snippet: Fig. 6 RO8191 reverses SH-4–54-mediated glioma cell death and pyroptosis. A, B Immunoblotting shows protein levels of NLRP3, GSDMD, N-GSDMD in control, SH-4–54, and SH-4–54 + RO8191 groups after SH-4–54 treatment. C Microscopic observation of the morphological effects of RO8191 on U87 and LN229 cells after SH-4–54 treatment. D, E EdU assay demonstrates proliferation capabilities in control, SH-4–54, and SH-4–54 + RO8191 groups. For A, B, data are presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, N.S. not significant by ANOVA.

Article Snippet: Antibodies were sourced from the following providers: Cell Signaling Technology (Danvers, Massachusetts, USA)—anti-EZH2 (1:1000), anti- STAT3 (1:1000), anti-phospho (p)-STAT3 (1:1000); Affinity Biologicals (Jiangsu, China)—anti-NLRP3 (1:1000); Abcam (Cambridge, MA, USA)— anti-GSDMD (1:1000), anti-cleaved N-terminal GSDMD (1:1000); Proteintech (Wuhan, China)—Beta Actin Recombinant antibody (1:20,000), IL-1 Beta Polyclonal antibody, IL-18 Polyclonal antibody; secondary antibodies (goat anti-rabbit and goat anti-mouse).

Techniques: Western Blot, Control, EdU Assay

Journal: Cell death discovery

Article Title: EZH2-STAT3 signaling pathway regulates GSDMD-mediated pyroptosis in glioblastoma.

doi: 10.1038/s41420-024-02105-0

Figure Lengend Snippet: Fig. 7 Schematic diagram of DZNep regulates pyroptosis in glioblastoma through the EZH2–STAT3 signaling pathway. In glioma cells, EZH2-related genes are closely associated with the cell cycle. EZH2 regulates the expression of the inflammasome NLRP3 via the STAT3 signaling pathway. By inhibiting STAT3, NLRP3 can be activated to cleave GSDMD into its N-GSDMD form. The cleaved GSDMD then translocates to the cell membrane to form pores, leading to apoptosis in glioma cells. This process is accompanied by the production of significant amounts of IL-1β and IL-18.

Article Snippet: Antibodies were sourced from the following providers: Cell Signaling Technology (Danvers, Massachusetts, USA)—anti-EZH2 (1:1000), anti- STAT3 (1:1000), anti-phospho (p)-STAT3 (1:1000); Affinity Biologicals (Jiangsu, China)—anti-NLRP3 (1:1000); Abcam (Cambridge, MA, USA)— anti-GSDMD (1:1000), anti-cleaved N-terminal GSDMD (1:1000); Proteintech (Wuhan, China)—Beta Actin Recombinant antibody (1:20,000), IL-1 Beta Polyclonal antibody, IL-18 Polyclonal antibody; secondary antibodies (goat anti-rabbit and goat anti-mouse).

Techniques: Expressing, Membrane

Journal: Frontiers in Immunology

Article Title: The role of gasdermin-mediated mitochondrial RNA release in amplifying secondary immune response during microbial infection

doi: 10.3389/fimmu.2025.1668763

Figure Lengend Snippet: GSDMD cleavage mediates mtRNA release and secondary inflammatory responses via the VISA pathway. (A, B) THP-1 cells primed with LPS and treated with Ng, and cells pretreated with VX-765 and LPS + Ng had their cytoplasmic RNA extracted and 2 μg/mL transfected into fresh THP-1 cells. qPCR analysis showed significantly higher IFN-β (A) and IL-6 (B) expression in fresh THP1 cells transfected from treated with LPS + Ng alone compared to VX-765-pretreated cells. Data are presented as mean ± SEM (****p < 0.0001, ***p < 0.001). (C, D) PM cells primed with LPS and treated with Ng, and cells pretreated with VX-765, then treated with LPS + Ng, had their cytoplasmic RNA extracted, and 2 μg/mL transfected into fresh PM cells. qPCR analysis showed significantly higher IFN-β (C) and IL-6 (D) expression in fresh PM cells transfected from treated with LPS + Ng alone compared to VX-765-pretreated cells. Data are presented as mean ± SEM (***p < 0.001, ****p < 0.0001). (E, F) WT PM cells and VISA -/- PM cells, primed with LPS and then treated with Ng, had their cytoplasmic RNA extracted and 2 μg/mL transfected into fresh WT PM cells and VISA − / − PM cells. qPCR analysis showed significantly higher IFN-β (E) and IL-6 (F) expression in fresh WT PM cells than in fresh VISA -/- PM cells. Data are presented as mean ± SEM (***p < 0.001, **p < 0.01). (G) Six WT mice (three peritoneal injected with 10 mg/kg LPS and three untreated) and three VISA -/- mice peritoneal injected with 10 mg/kg LPS. Western blot analysis of lung tissues showed no GSDMD cleavage or pTBK1 expression in untreated WT mice. In contrast, LPS-injected WT mice exhibited both GSDMD cleavage and pTBK1 expression, whereas LPS-injected VISA -/- mice displayed GSDMD cleavage but no pTBK1 expression. (H) THP-1 cells were primed with LPS alone, cells primed with LPS were then treated with Ng, and cells pretreated with VX-765, followed by LPS + Ng treatment, and their culture media was collected. RNA extraction and qPCR analysis revealed higher expression of mtRNA (ND5, ND6, and CYTB) in the extracellular space of cells treated with LPS + Ng alone compared to VX-765-pretreated cells. Data are presented as mean ± SEM (**p < 0.01, **p < 0.01, *p < 0.05). (I) PM cells primed with LPS only, primed with LPS, then treated with Ng, and cells pretreated with VX-765, followed by LPS + Ng treatment, had their culture media collected. RNA extraction and qPCR analysis revealed higher expression of mtRNA (ND1, COX1, and CYTB) in the extracellular space of cells treated with LPS + Ng alone compared to VX-765-pretreated cells. Data are presented as mean ± SEM (**p < 0.01, ****p < 0.0001, *p < 0.05), all experiments were repeated three times, and representative experiments are shown.

Article Snippet: Monoclonal rabbit antibodies against GSDME (ab215191) were purchased from Abcam at a dilution of 1:2000, and monoclonal rabbit antibodies against GSDMD (97558) from Cell Signaling Technology at a dilution of 1:1000, monoclonal rabbit antibodies against phospho-TBK1 (ab109272) from Abcam at a dilution of 1:2000, monoclonal rabbit antibody against Caspase-1 (3866) purchased from Cell Signaling Technology at a dilution of 1:1000, monoclonal rabbit antibody against NLRP3 (15101) sourced from Cell Signaling Technology at a dilution of 1:1000 and monoclonal antibody against VDAC1 (55259-1-AP) from Proteintech.

Techniques: Transfection, Expressing, Injection, Western Blot, RNA Extraction

Journal: Frontiers in Immunology

Article Title: The role of gasdermin-mediated mitochondrial RNA release in amplifying secondary immune response during microbial infection

doi: 10.3389/fimmu.2025.1668763

Figure Lengend Snippet: Gasdermin D and E mediated mtRNA release and VISA pathway activation induce secondary inflammatory response. Cells treated with LPS and Ng trigger the activation of the NLRP3 pathway, leading to the recruitment of caspase-1, which in turn cleaves GSDMD . In contrast, cells infected with VSV activate the PKR pathway and caspase-3, resulting in the cleavage of GSDME ( , ). Our findings indicate that the N-terminal fragments of both GSDMD and GSDME facilitate mitochondrial membrane permeabilization and the release of mtRNA into the cytoplasm. This mtRNA is subsequently recognized by the double-stranded RNA sensors RIG-I and MDA5, prompting their activation . Activated RIG-I interacts with VISA (MAVS) on the mitochondrial membrane , initiating a signaling cascade that involves various kinases, including TBK-1, IKKϵ, IKKα, and IKKβ ( , ). This cascade culminates in the phosphorylation and activation of key transcription factors, notably IRF-3/7 and NF-κB. These activated transcription factors then translocate to the nucleus to drive the expression of type I interferons (IFNs) and an array of pro-inflammatory cytokines ( , ). Furthermore, the N-terminal fragments of Gasdermin D and E contribute to the formation of pores in the plasma membrane, facilitating the release of mtRNA into the extracellular space due to the permeabilization induced by GSDMD and GSDME.

Article Snippet: Monoclonal rabbit antibodies against GSDME (ab215191) were purchased from Abcam at a dilution of 1:2000, and monoclonal rabbit antibodies against GSDMD (97558) from Cell Signaling Technology at a dilution of 1:1000, monoclonal rabbit antibodies against phospho-TBK1 (ab109272) from Abcam at a dilution of 1:2000, monoclonal rabbit antibody against Caspase-1 (3866) purchased from Cell Signaling Technology at a dilution of 1:1000, monoclonal rabbit antibody against NLRP3 (15101) sourced from Cell Signaling Technology at a dilution of 1:1000 and monoclonal antibody against VDAC1 (55259-1-AP) from Proteintech.

Techniques: Activation Assay, Infection, Membrane, Phospho-proteomics, Expressing, Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: The role of gasdermin-mediated mitochondrial RNA release in amplifying secondary immune response during microbial infection

doi: 10.3389/fimmu.2025.1668763

Figure Lengend Snippet: GSDMD inhibition modulates cytokine production in LPS and Ng responses. (A, B) THP1 cells were primed with 100 ng/mL LPS for 2 hours and activated with 5 μM/mL nigericin (Ng) for one hour, with qPCR analysis demonstrating elevated expression of IFN-β (A) and IL-6 (B) . An unpaired t-test determined statistical significance. Data represent ± SEM (**p < 0.01, ***p < 0.001). (C, D) mouse peritoneal macrophages (PM cells) primed with 100 ng/mL LPS for 2 hours, treated with 5 μM/mL Ng for one hour, qPCR analysis demonstrating elevated expression of IFN-β (C) and IL-6 (D) , an unpaired t-test determined statistical significance. Data represent ± SEM (**p < 0.01, **p < 0.01). (E, F) THP-1 and PM cells primed with 100 ng/mL LPS alone exhibited reduced NLRP3 activation, caspase-1, and GSDMD cleavage. In contrast, cells primed with LPS and activated with 5 μM Ng showed enhanced NLRP3 activation, caspase-1, and GSDMD cleavage, leading to TBK1 phosphorylation. Cleaved caspase-1 protein (p20) was isolated from the supernatant (SN), and pTBK1 protein expression levels were quantified using ImageJ; an unpaired t-test determined statistical significance. Data represent ± SEM (***p < 0.001, ****p < 0.0001). (G, H) THP1 cells primed with 100 ng/mL LPS alone, cells treated with LPS + Ng, cells pretreated with 20 μM VX-765 for 2 hours and then treated with LPS + Ng, qPCR analysis showing decreased IFN-β (E) and IL-6 (F) expression in VX-765 pretreated THP-1 cells. Data represent mean ± SEM (*p < 0.05, ***p < 0.001). (I, J) PM cells primed with LPS alone, cells treated with LPS + Ng, cells pretreated with 20 μM VX-765 for 2 hours and then treated with LPS + Ng, qPCR analysis showing decreased IFN-β (G) and IL-6 (H) expression in VX-765 pretreated PM cells. Data represent mean ± SEM (**p < 0.01, *p < 0.05). (K, L) Western blot analysis of THP1 and PM cells showed no GSDMD cleavage and TBK1 phosphorylation in LPS-primed cells alone and cells pretreated with 20 μM VX-765 for 2 hours followed by LPS + Ng treatment. In contrast, LPS + Ng treatment induced GSDMD cleavage and elevated TBK1 phosphorylation, pTBK1 protein expression levels were quantified using ImageJ; an unpaired t-test determined statistical significance. Data represent ± SEM (****p < 0.0001, ***p < 0.001). (M, N) THP1 cells primed with LPS alone, cells primed with LPS, then treated with Ng, cells primed with LPS, then treated with 100 μM/mL dimethyl fumarate (DMF) for 2 hours and then treated with Ng, qPCR analysis showing decreased IFN-β (K) and IL-6 (L) expression in DMF treated THP1 cells. Data represent mean ± SEM (***p < 0.001, ****p < 0.0001). (O, P) PM cells primed with LPS alone, cells primed with LPS and then treated with Ng, cells primed with LPS, then treated with 100 μM/mL DMF for 2 hours and then treated with Ng, qPCR analysis showing decreased IFN-β (M) and IL-6 (N) expression in DMF treated PM cells. Data represent mean ± SEM (**p < 0.01, **p < 0.01). (Q, R) Western blot analysis showed that LPS priming, followed by Ng treatment, induced GSDMD cleavage and elevated TBK1 phosphorylation in THP-1 and PM cells. However, cells primed with LPS, treated with DMF, and treated with Ng inhibited GSDMD cleavage and reduced TBK1 phosphorylation, pTBK1 protein expression levels were quantified using ImageJ; an unpaired t-test determined statistical significance. Data represent ± SEM (****p < 0.0001, ***p < 0.001), all experiments were repeated three times, and representative experiments are shown.

Article Snippet: Monoclonal rabbit antibodies against GSDME (ab215191) were purchased from Abcam at a dilution of 1:2000, and monoclonal rabbit antibodies against GSDMD (97558) from Cell Signaling Technology at a dilution of 1:1000, monoclonal rabbit antibodies against phospho-TBK1 (ab109272) from Abcam at a dilution of 1:2000, monoclonal rabbit antibody against Caspase-1 (3866) purchased from Cell Signaling Technology at a dilution of 1:1000, monoclonal rabbit antibody against NLRP3 (15101) sourced from Cell Signaling Technology at a dilution of 1:1000 and monoclonal antibody against VDAC1 (55259-1-AP) from Proteintech.

Techniques: Inhibition, Expressing, Activation Assay, Phospho-proteomics, Isolation, Western Blot

Journal: Frontiers in Immunology

Article Title: The role of gasdermin-mediated mitochondrial RNA release in amplifying secondary immune response during microbial infection

doi: 10.3389/fimmu.2025.1668763

Figure Lengend Snippet: GSDMD as key drivers of mitochondrial dysfunction: (A) THP1 cells primed with LPS alone, cells primed with LPS, then treated with Ng, cells pretreated with VX-765, then treated with LPS + Ng. Subjected to mitochondrial Isolation, Western blot analysis of GSDMD cleavage was observed in both cytoplasmic and mitochondrial fractions of LPS + Ng-treated cells; no GSDMD cleavage was observed in cells pretreated with VX-765 or those primed with LPS alone. (B) PM cells primed with LPS, cells primed with LPS, then treated with Ng, cells pretreated with VX-765, then treated with LPS + Ng. Subjected to mitochondrial Isolation, Western blot analysis of GSDMD cleavage was observed in both cytoplasmic and mitochondrial fractions of LPS + Ng-treated cells; no GSDMD cleavage was observed in cells pretreated with VX-765 or those primed with LPS alone. (C) THP1 cells primed with LPS alone, cells primed with LPS, then treated with Ng, cells pretreated with VX-765, then treated with LPS + Ng. Cells stained with JC-1 dye, LPS + Ng alone displayed a higher proportion of JC-1 monomers and fewer aggregates, in contrast to THP-1 cells primed with LPS alone and cells pretreated with VX-765. (D, E) THP1 cells were primed with LPS alone, primed with LPS, then treated with Ng, pretreated with VX-765, then treated with LPS + Ng treatment, and mitochondrial damage was assessed using JC-1 staining. Flow cytometry analysis confirmed the results observed by inverted microscopy; the histogram shows the percentage. (F) PM cells primed with LPS alone, cells primed with LPS, then treated with Ng, cells pretreated with VX-765, then treated with LPS + Ng. Cells stained with JC-1 dye, LPS + Ng alone displayed a higher proportion of JC-1 monomers and fewer aggregates, in contrast to PM cells primed with LPS alone and cells pretreated with VX-765. (G, H) PM cells were primed with LPS alone, primed with LPS, then treated with Ng, pretreated with VX-765, then treated with LPS + Ng treatment, and mitochondrial damage was assessed using JC-1 staining. Flow cytometry analysis confirmed the results observed by inverted microscopy; the histogram shows the percentage, all experiments were repeated three times, and representative experiments are shown. The histogram shows the percentage of Flow Cytometry analysis, with statistical significance determined by an unpaired t-test. Data represent ± SEM (***p < 0.001, ****p < 0.0001).

Article Snippet: Monoclonal rabbit antibodies against GSDME (ab215191) were purchased from Abcam at a dilution of 1:2000, and monoclonal rabbit antibodies against GSDMD (97558) from Cell Signaling Technology at a dilution of 1:1000, monoclonal rabbit antibodies against phospho-TBK1 (ab109272) from Abcam at a dilution of 1:2000, monoclonal rabbit antibody against Caspase-1 (3866) purchased from Cell Signaling Technology at a dilution of 1:1000, monoclonal rabbit antibody against NLRP3 (15101) sourced from Cell Signaling Technology at a dilution of 1:1000 and monoclonal antibody against VDAC1 (55259-1-AP) from Proteintech.

Techniques: Isolation, Western Blot, Staining, Flow Cytometry, Inverted Microscopy

Journal: Nature Chemical Biology

Article Title: DPP8 and DPP9 inhibition induces pro-caspase-1-dependent monocyte and macrophage pyroptosis

doi: 10.1038/nchembio.2229

Figure Lengend Snippet: Figure 5 | GSDMD is cleaved after DPP8/9 inhibition and contributes to cell death. (a) GSDMD is cleaved in THP-1 macrophages after treatment with Val-boroPro (VbP) or 1G244 for 24 h, but CASP-3, CASP-7 and PARP are not. FL, full-length; CL, cleaved. Asterisks denote non-specific bands. (b) GSDMD is cleaved after Val-boroPro treatment of THP-1, RAW 264.7, J774 and primary human PBMCs, as determined by immunoblotting. RAW 264.7 do not cleave Gsdmd after LPS plus nigericin treatment, as expected. Nig, nigericin. (c,d) The pyroptotic response in GSDMD-deficient THP-1 macrophages, which were validated by immunoblotting (c), was delayed but not entirely prevented (d). Full gel images for a–c are shown in Supplementary Figure 12. In d, data represent mean ± s.e.m. of three biological replicates.

Article Snippet: Antibodies used include: human caspase-1 (no. 2225, Cell Signaling Technology), mouse caspase-1 (clone Casper-1, Adipogen), caspase-3 (clone 8G10, Cell Signaling Technology), human caspase-4 (clone 4B9, Santa Cruz), human caspase-5 (clone D3G4W, Cell Signaling Technology), caspase-7 (clone D2Q3L, Cell Signaling Technology), human IL-1β (Clone 2805, R&D Systems), mouse IL-1β (clone D4T2D, Cell Signaling Technology), IL-1α (no. AF-200, R&D Systems), IL-18 (no. AF2548, R&D Systems), GAPDH (clone 14C10, Cell Signaling Technology), DPP7 (Clone 398024, R&D Systems), DPP8 (ab42076, Abcam), DPP9 (ab42080, Abcam), PARP (no. 9542, Cell Signaling Technology), GSDMD (NBP2-33422, Novus Biologicals), DPP4 (no. 11D7, GeneTex), FAP (ABT11, Millipore) and SCPEP1 (SAB2700267, Sigma).

Techniques: Inhibition, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: NLRP3-mediated epithelial pyroptosis involved in airway hyperresponsiveness in combined eosinophilic and neutrophilic asthma

doi: 10.1186/s12964-026-02706-5

Figure Lengend Snippet: Evidence of cell pyroptosis existed in EOS + NEU + inflammation asthma. a Protein levels of NLRP3, cleaved caspase-1 and GSDMD-NT were upregulated in HBE after 24 h stimulation with IL-13 and IL-17 A. b , c IL-1β and LDH levels in cell culture supernatants were measured ( n = 6 per group). d Scanning electron microscopy revealed pore formation on the surface of HBEs stimulated with IL-13 and IL-17 A, scale bar, 5 μm. e , f Expression of GSDMD-NT and cleaved caspase-1 was elevated in lung tissues of mice exposed to OVA and ozone, accompanied by the dysfunction of cell junctions. Scale bar, 500 nm. g - i mRNA expression levels of caspase-1, GSDMD and IL-1β were increased. Data were expressed as mean ± SD. Abbreviations: Ctrl, control; EOS, eosinophil; GSDMD, Gasdermin D; GSDMD-NT, Gasdermin D-N-terminal; HBE, human bronchial epithelial cells; IL, interleukin; LDH, lactate dehydrogenase; NEU, neutrophil; NLRP3, NOD-like receptor protein 3; OVA, ovalbumin; SD, standard deviation

Article Snippet: For GSDMD inhibition, disulfiram (50 mg/kg in corn oil; HY-B0240, MedChemExpress, Shanghai, China) or vehicle was administered via intraperitoneal injection before each OVA challenge, following established protocols [ , ].

Techniques: Cell Culture, Electron Microscopy, Expressing, Control, Standard Deviation

Journal: Cell Communication and Signaling : CCS

Article Title: NLRP3-mediated epithelial pyroptosis involved in airway hyperresponsiveness in combined eosinophilic and neutrophilic asthma

doi: 10.1186/s12964-026-02706-5

Figure Lengend Snippet: NLRP3 inhibition alleviates AHR and suppresses GSDMD activation. a , b AHR was reduced in NLRP3 −/− mice after OVA and ozone exposure. c - g HE and PAS staining revealed attenuated airway inflammation and mucus hypersecretion in NLRP3 −/− mice. h Immunofluorescence showed decreased GSDMD-NT expression in lung tissues, scale bars 100 µm. i - l Inflammatory cytokine levels and caspase-1 in BALF or lung tissue were measured using ELISA. m Treatment with MCC950 reduced the expression of NLRP3, cleaved caspase-1 and GSDMD-NT in HBE. n Immunofluorescence demonstrated changes in NLRP3 and GSDMD-NT expression in HBEs treated with MCC950, scale bars 200 µm. Data were expressed as mean ± SD. Abbreviations : AHR Airway hyperresponsiveness, BALF Bronchoalveolar lavage fluid, Ctrl , Control, DAPI 4’,6-diamidino-2-phenylindole, ELISA Enzyme-linked immunosorbent assay, GSDMD Gasdermin D, GSDMD-NT Gasdermin D-N-terminal, HBE Human bronchial epithelial cells, HE Haematoxylin and eosin, NLRP3 NOD-like receptor protein 3, OVA Ovalbumin, PAS Periodic acid-Schiff

Article Snippet: For GSDMD inhibition, disulfiram (50 mg/kg in corn oil; HY-B0240, MedChemExpress, Shanghai, China) or vehicle was administered via intraperitoneal injection before each OVA challenge, following established protocols [ , ].

Techniques: Inhibition, Activation Assay, Staining, Immunofluorescence, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Cell Communication and Signaling : CCS

Article Title: NLRP3-mediated epithelial pyroptosis involved in airway hyperresponsiveness in combined eosinophilic and neutrophilic asthma

doi: 10.1186/s12964-026-02706-5

Figure Lengend Snippet: Inhibition of GSDMD alleviates mixed airway inflammation and AHR. a - d Lung function variables improved following disulfiram treatment ( n = 6 per group). e , f Disulfiram significantly reduced AHR in EOS + NEU + asthmatic mice. g - i HE and PAS staining analysis demonstrated reduced airway inflammation and mucus secretion after GSDMD inhibition. j - l Levels of inflammatory cytokines in BALF were decreased following disulfiram treatment; ( g , h ) HBEs were transfected with GSDMD small interfering RNA (siRNA) for 24 h and were then treated with IL-13/IL17A for another 24 h. The expression of IL-1β was studied using ELISA analysis. Data were expressed as mean ± SD. Abbreviations: AHR Airway hyperresponsiveness, BALF Bronchoalveolar lavage fluid, ELISA Enzyme-linked immunosorbent assay, GAPDH Glyceraldehyde-3-phosphate dehydrogenase, GSDMD Gasdermin D, HBE Human bronchial epithelial cells, HE Haematoxylin and eosin, IL Interleukin, OVA Ovalbumin

Article Snippet: For GSDMD inhibition, disulfiram (50 mg/kg in corn oil; HY-B0240, MedChemExpress, Shanghai, China) or vehicle was administered via intraperitoneal injection before each OVA challenge, following established protocols [ , ].

Techniques: Inhibition, Staining, Transfection, Small Interfering RNA, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Virus Research

Article Title: Role of heat shock protein 90 as an antiviral target for swine enteric coronaviruses

doi: 10.1016/j.virusres.2023.199103

Figure Lengend Snippet: SADS-CoV infection triggers GSDMD-mediated pyroptosis, which was reduced by inhibition of Hsp90. (A) Macrophages were infected with SADS-CoV at MOI=10. Viral load was detected in cell lysate or supernatant by one-step qRT-PCR at the indicated hpi. (B) Macrophages were infected with SADS-CoV at MOI=10 for 24 h in the presence 17-DMAG. Cells were lysed and levels of pro-caspase-1, GSDMD, cleaved GSDMD, Hsp70, SADS-CoV N and β-actin were determined by western blot. (C) The protein level of GSDMD, cleaved GSDMD and pro-caspase-1 were quantified by immunoblot scanning and normalized with respect to β-actin. (D) qRT-PCR of TNF-α, IL-10, IL-8, IL-1β and IL-6 mRNA levels in Macrophages infected with SADS-CoV (MOI of 10) for 24 h in the presence 17-DMAG. The relative expression of target genes was normalized to GAPDH rRNA; **: P ≤ 0.01; ***: P ≤ 0.001; N.S.: not significant.

Article Snippet: Myc-tag (#2278), Hsp90α (#8165) rabbit monoclonal antibody, Hsp90β antibody (#5087), Hsp70 antibody (#4872), GSDMD antibody (#39,754) and anti-β-actin (#3700) mouse monoclonal antibody were purchased from Cell Signaling.

Techniques: Infection, Inhibition, Quantitative RT-PCR, Western Blot, Expressing

Journal: Science advances

Article Title: Targeting pyroptosis with nanoparticles to alleviate neuroinflammatory for preventing secondary damage following traumatic brain injury.

doi: 10.1126/sciadv.adj4260

Figure Lengend Snippet: Fig. 4. Inhibitory effect of C-β-LG/DSF on pyroptosis after TBI. (A) The schematic diagram illustrates the inhibition of pyroptosis by DSF. After the uptake of C-β-LG/DSF by cells, DSF inhibits the aggregation of GSDMD N-terminal pores on the cell membrane surface. Furthermore, experimental results demonstrated that the expression of GSDMD, GSDMD-N, caspase-1, and other related proteins was reduced after treatment with C-β-LG/DSF. (B) Immunoblotting of GSDMD, GSDMD-N, and caspase-1, caspase-1 p10, caspase-1 p20 in neuron-ICR cells with sham, TBI, DSF, and C-β-LG/DSF for 24 hours. (C to E) Levels of (C) IL-1β, (D) IL-18, and (E) LDH in injured neuron-ICR as detected by ELISA 12 hours after TBI. (F) Immunohistochemical staining was used to observe the expression of GSDMD in injured tissues of TBI model mice 3 days after different drugs treatment. Scale bars, 50 μm. (G) Quantitative analysis of GSDMD-positive cells. (H to J) Levels of (H) IL-1β, (I) IL-18, and (J) LDH in injured tissues as detected by ELISA 3 days after TBI. (K) Immunoblotting of GSDMD, GSDMD-N, caspase-1, caspase-1 p20, and caspase-1 p10 in injured tissue with sham, TBI, DSF, β-LG/DSF, and C-β-LG/ DSF for 24 hours. Data were expressed as means ± SD (n = 5). For (C) to (E) and (G) to (J), statistical analysis was calculated via one-way ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: Primary antibodies used for immunoblot were listed as follows: anti– caspase- 1 p20 (Santa Cruz Biotechnology, sc- 398715; 1:1000), anti– cleaved GSDMD (Cell Signaling Technology, #10137S; 1:1000), anti–caspase- 1 p10 (GeneTex, GTX134551; 1:1000), and anti- GAPDH (Proteintech, 60004- 1- Ig; 1:1000).

Techniques: Inhibition, Membrane, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Dual NLRC4 and non-canonical inflammasome signaling drives human GSDMD-mediated killing of Shigella flexneri independently of bacterial cardiolipin

doi: 10.64898/2026.01.11.698901

Figure Lengend Snippet: (A) Lack of impact of IFNγ priming on cell death of S. flexneri -infected macrophages. Cell death measured as lactate dehydrogenase release. (B) Schematic of pyroptosis with indicated sites of action of inhibitors of gasdermin D (GSDMD) N-terminal domain (NT) pore formation (DSF, disulfiram) and of ninjurin-1 (NINJ1) oligomerization and plasma membrane rupture (PMR; glycine). CASP1/4, caspase-1 and/or -4. (C) Intracellular S. flexneri upon inhibition of lytic cell death. Macrophages, primed or not primed with IFNγ, were treated with glycine in the absence of gentamicin. (D) Inhibition of S. flexneri infection-induced release of activated caspase-4 (CASP4 p32) and activated caspase-1 (CASP1 p20) by disulfiram. Representative immunoblots. (E) Intracellular S. flexneri upon inhibition of GSDMD plasma membrane pore formation. Macrophages, primed or not primed with IFNγ, were treated with disulfiram in the absence of gentamicin. (F) IFNγ mediated restriction is not due to bacterial loss in cell culture supernatant or detached macrophages. S. flexneri in cell culture supernatants and washes (which contain detached macrophages and released bacteria), and in attached macrophages. Samples collected at indicated times during infection. Graphed are bacterial counts combined for supernatants, washes, and attached cells. Data represent the mean ± SEM. ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant, by two-tailed unpaired Student’s t-test (F) or ordinary two-way ANOVA (A, C, E).

Article Snippet: Antibodies used for immunoblotting were as follows: caspase-1 (Abcam, ab207802) rabbit monoclonal antibody at 0.5 μg/mL (1:1000), GBP1 (Abcam, ab131255) rabbit monoclonal antibody at 0.25 μg/mL (1:5000), caspase-4 (Santa Cruz, sc-56056) and GSDMD (Santa Cruz, sc-81868) mouse monoclonal antibodies at 0.5 μg/mL (1:200).

Techniques: Infection, Clinical Proteomics, Membrane, Inhibition, Western Blot, Cell Culture, Bacteria, Two Tailed Test

Journal: bioRxiv

Article Title: Dual NLRC4 and non-canonical inflammasome signaling drives human GSDMD-mediated killing of Shigella flexneri independently of bacterial cardiolipin

doi: 10.64898/2026.01.11.698901

Figure Lengend Snippet: (A) Rescue of intracellular S. flexneri in the absence of GSDMD , with or without IFNγ priming. (B) Schematic of S. flexneri activation of caspase-1 inflammasomes in human macrophages. ASC, apoptosis-associated speck-like protein containing a CARD; CASP1, caspase-1; NT-GSDMD, N-terminal pore-forming domain of GSDMD. (C) Rescue of intracellular S. flexneri in the absence of CASP1 , with or without IFNγ priming. (D) Caspase-1 is required for S. flexneri infection-induced activation of GSDMD (GSDMD p30). Unprimed macrophages. (E-F) Lack of impact of NLRP3 inhibition by MCC950 on numbers of intracellular S. flexneri in macrophages, with or without IFNγ priming (E) and on infection-induced caspase-1 activation (F). Positive control: treatment with NLRP3 agonist nigericin. (G) Reduced processing of caspase-1 (CASP1 p20) and GSDMD (GSDMD p30) during S. flexneri infection in the absence of NLRC4 . (H) Rescue in NLRC4 -/- macrophages of intracellular S. flexneri in the absence but not the presence of IFNγ priming. Immunoblots are representative (D, F-G). Data represent the mean ± SEM. * p < 0.05, **** p < 0.0001, ns, not significant, by ordinary two-way ANOVA.

Article Snippet: Antibodies used for immunoblotting were as follows: caspase-1 (Abcam, ab207802) rabbit monoclonal antibody at 0.5 μg/mL (1:1000), GBP1 (Abcam, ab131255) rabbit monoclonal antibody at 0.25 μg/mL (1:5000), caspase-4 (Santa Cruz, sc-56056) and GSDMD (Santa Cruz, sc-81868) mouse monoclonal antibodies at 0.5 μg/mL (1:200).

Techniques: Activation Assay, Infection, Inhibition, Positive Control, Western Blot

Journal: bioRxiv

Article Title: Dual NLRC4 and non-canonical inflammasome signaling drives human GSDMD-mediated killing of Shigella flexneri independently of bacterial cardiolipin

doi: 10.64898/2026.01.11.698901

Figure Lengend Snippet: (A) Schematic of S. flexneri activation of inflammasomes that depend on caspase-4 (CASP4). In macrophages, S. flexneri infection activates GSDMD via NLRP11. 9 In epithelial cells, caspase-4 is activated by guanylate-binding protein 1 (GBP1) recognition of LPS, which S. flexneri inhibits. – NT-GSDMD, N-terminal pore-forming domain of GSDMD. (B-C) The absence of CASP4 (B) or CASP4 and CASP1 (C) impacts intracellular S. flexneri in the presence but not absence of IFNγ-priming. (D) Levels of GBP1 in macrophages infected with WT or Δ ipaH9.8 S. flexneri . (E) Lack of impact of GBP1 on restriction of S. flexneri . GBP1 depletion by RNA interference (siRNA). scRNA, scrambled RNA interference control. (F) Lack of significant impact of NLRP11 on intracellular S. flexneri in macrophages, with or without IFNγ priming. (G) IFNγ priming of S. flexneri infection is associated with increased levels of cellular caspase-4 (CASP4) and caspase-1 (CASP1), increased release of cleaved CASP4 (CASP4 p32) and cleaved CASP1 (CASP1 p20) into the cell culture supernatants, and increased cellular cleavage of GSDMD to release the pore-forming domain (GSDMD p30). Shorter, shorter exposure. Immunoblots are representative (D-E, G). Data represent the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant by ordinary two-way ANOVA.

Article Snippet: Antibodies used for immunoblotting were as follows: caspase-1 (Abcam, ab207802) rabbit monoclonal antibody at 0.5 μg/mL (1:1000), GBP1 (Abcam, ab131255) rabbit monoclonal antibody at 0.25 μg/mL (1:5000), caspase-4 (Santa Cruz, sc-56056) and GSDMD (Santa Cruz, sc-81868) mouse monoclonal antibodies at 0.5 μg/mL (1:200).

Techniques: Activation Assay, Infection, Binding Assay, Control, Cell Culture, Western Blot

Journal: bioRxiv

Article Title: Dual NLRC4 and non-canonical inflammasome signaling drives human GSDMD-mediated killing of Shigella flexneri independently of bacterial cardiolipin

doi: 10.64898/2026.01.11.698901

Figure Lengend Snippet: (A) Replication of S. flexneri in GSDMD -/- macrophages. Numbers of S. flexneri in cell culture supernatants and washes (containing detached macrophages and released bacteria) and attached macrophages, at the indicated infection times. Graphed are bacterial counts combined for supernatants, washes, and attached cells. (B) Infected GSDMD -/- macrophages harbor significantly more S. flexneri per cell than infected WT macrophages. Left panel, immunofluorescence at 2 hours of infection using Hoechst (blue) and antibody to S. flexneri (green) (representative images). Right graph, numbers of bacteria per cell. Each symbol in the scatter blot represents one infected cell. Minimum of 480 cells (160 per biological replicate) were scored for each condition. Scale bar: 10 µm. (C) Lack of impact of GSDMD on survival of intravacuolar S. flexneri Δ ipaC . (D) Enhanced GSDMD-dependent killing of intracellular S. flexneri in macrophages treated with extracellular LPS and nigericin. (E) Killing of intracellular S. flexneri upon infection of HEK293T cells expressing full-length GSDMD (FL GSDMD) or the GSDMD N-terminal pore-forming domain (NT-GSDMD). Survival is calculated using bacterial counts combined for supernatants, washes, and attached cells. Data represent the mean ± SEM (A, C-E) or the median (B). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant, by two-tailed unpaired Student’s t-test (A-B) or ordinary one- (E) or two-way (C-D) ANOVA.

Article Snippet: Antibodies used for immunoblotting were as follows: caspase-1 (Abcam, ab207802) rabbit monoclonal antibody at 0.5 μg/mL (1:1000), GBP1 (Abcam, ab131255) rabbit monoclonal antibody at 0.25 μg/mL (1:5000), caspase-4 (Santa Cruz, sc-56056) and GSDMD (Santa Cruz, sc-81868) mouse monoclonal antibodies at 0.5 μg/mL (1:200).

Techniques: Cell Culture, Bacteria, Infection, Immunofluorescence, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Dual NLRC4 and non-canonical inflammasome signaling drives human GSDMD-mediated killing of Shigella flexneri independently of bacterial cardiolipin

doi: 10.64898/2026.01.11.698901

Figure Lengend Snippet: (A) Lack of impact of cardiolipin on GSDMD-mediated killing, with or without IFNγ priming. WT and GSDMD -/- macrophages infected with WT or cardiolipin mutant (Δ clsA ) of S. flexneri . (B) S. flexneri Δ clsA -infected GSDMD -/- macrophages harbor significantly more bacteria per cell than S. flexneri Δ clsA -infected WT macrophages. Left panel, immunofluorescence at 2 hours of infection using Hoechst (blue) and antibody to S. flexneri (green) (representative images). Right graph, numbers of bacteria per cell. Each symbol in the scatter blot represents one infected cell. Minimum of 420 cells (140 per biological replicate) were scored for each condition. Scale bar: 10 µm. (C) Enhanced killing of intracellular S. flexneri Δ clsA in macrophages treated with extracellular LPS and NLRP3 agonist nigericin. (D) Killing of intracellular S. flexneri Δ clsA upon infection of HEK293T cells expressing full-length GSDMD (FL GSDMD) or GSDMD N-terminal pore-forming domain (NT-GSDMD). Survival is calculated using bacterial counts combined for supernatants, washes, and attached cells. (E) Lack of significant differences in survival between WT and Δ clsA S. flexneri upon infection of HEK293T cells expressing indicated GSDMD constructs. For each bacterial strain, survival is calculated as compared to HEK293T cells transfected with the empty vector. (F) Colocalization of GSDMD and S. flexneri . Immunofluorescence using antibodies to GSDMD (red) and S. flexneri (green). Unprimed WT and GSDMD -/- macrophages infected with WT or Δ clsA S. flexneri for 50 mins. Representative images. Data represent the mean ± SEM (A, C-D) or median (B). * p < 0.05, ** p < 0.01, **** p < 0.0001, ns, not significant, by two-tailed unpaired Student’s t-test (B-C) or ordinary one- (D) or two-way (A and E) ANOVA.

Article Snippet: Antibodies used for immunoblotting were as follows: caspase-1 (Abcam, ab207802) rabbit monoclonal antibody at 0.5 μg/mL (1:1000), GBP1 (Abcam, ab131255) rabbit monoclonal antibody at 0.25 μg/mL (1:5000), caspase-4 (Santa Cruz, sc-56056) and GSDMD (Santa Cruz, sc-81868) mouse monoclonal antibodies at 0.5 μg/mL (1:200).

Techniques: Infection, Mutagenesis, Bacteria, Immunofluorescence, Expressing, Construct, Transfection, Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: Dual NLRC4 and non-canonical inflammasome signaling drives human GSDMD-mediated killing of Shigella flexneri independently of bacterial cardiolipin

doi: 10.64898/2026.01.11.698901

Figure Lengend Snippet: (A) First contact of macrophages with pathogens occurs in the absence of IFNγ priming. In this setting, S. flexneri is recognized by NLRC4, which leads to processing and activation of caspase-1 (CASP1) and downstream GSDMD. GSDMD targets and kills intracellular S. flexneri . (B) Once the innate immune system is activated, macrophages are primed with IFNγ, leading to increased caspase-4 (CASP4) and caspase-1 (CASP1) and enhanced activation of GSDMD, which increases killing of intracellular S. flexneri . In both cases, GSDMD kills S. flexneri independently of bacterial cardiolipin and host cell lysis.

Article Snippet: Antibodies used for immunoblotting were as follows: caspase-1 (Abcam, ab207802) rabbit monoclonal antibody at 0.5 μg/mL (1:1000), GBP1 (Abcam, ab131255) rabbit monoclonal antibody at 0.25 μg/mL (1:5000), caspase-4 (Santa Cruz, sc-56056) and GSDMD (Santa Cruz, sc-81868) mouse monoclonal antibodies at 0.5 μg/mL (1:200).

Techniques: Activation Assay, Lysis

Journal: Journal of Cellular and Molecular Medicine

Article Title: New mechanism of nerve injury in Alzheimer’s disease: β‐amyloid‐induced neuronal pyroptosis

doi: 10.1111/jcmm.15439

Figure Lengend Snippet: Effects of Aβ 1‐42 on pyroptosis in MCNs (n = 3). A, Results on relative uptake rate of PI: The relative uptake rate of PI was up‐regulated in the Aβ 1‐42 group with increasing time, compared with the control group, * P < .05; compared with the LPS + Nigericin group (positive control), * P < .05. Aβ 1‐42 treatment increased the opening of membrane pores in MCNs. B, Results of LDH on cytotoxicity: Aβ 1‐42 treatment up‐regulated the release of LDH in MCNs, resulting in cytotoxicity. Comparison between groups, * P < .05. C, Results of GSDMD mRNA expression: Aβ 1‐42 intervention up‐regulated the mRNA expression of GSDMD, while the mRNA expression of GSDMD was low in the control, indicating that Aβ 1‐42 promoted mRNA transcription. Comparison between groups, * P < .05. D, Results of PI and Hoechst 33 258 staining in MCNs: The number of positive‐staining cells was relatively less in the control group, while the number of positive‐staining cells in the LPS + Nigericin positive group was significantly increased, indicating the increased number of pyroptotic cells. The number of positive cells was also increased in the Aβ 1‐42 group, suggesting that Aβ 1‐42 ‐induced pyroptosis. E, Results on IF staining of GSDMD: The IF staining of GSDMD was relatively weak in control group, and the IF staining of GSDMD was significantly stronger in LPS + Nigericin and Aβ 1‐42 groups compared to that in control, indicating the increased expression of GSDMD

Article Snippet: To determine whether Aβ 1‐42 induced pyroptosis activating GSDMD oligomerization, the p30‐GSDMD oligomerization inhibitor, necrosulfonamide (NSA) (MCE, Shanghai, China) was used for intervention.

Techniques: Control, Positive Control, Membrane, Comparison, Expressing, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: New mechanism of nerve injury in Alzheimer’s disease: β‐amyloid‐induced neuronal pyroptosis

doi: 10.1111/jcmm.15439

Figure Lengend Snippet: Effects of Aβ1‐42 on inflammatory factor release and expression of pyroptosis‐related proteins in MCNs (n = 3). A‐C, Expression levels of inflammatory factors, including IL‐6, IL‐1β and TNF‐α in cell culture medium. The expression of these inflammatory factors was not significantly changed and remained low over time in the control group, while that was up‐regulated over time in the LPS + Nigericin and Aβ1‐42 groups, indicating increased cell permeability and enhanced release of inflammatory factors. Comparison between Aβ1‐42 group and control group, * P < .05; Comparison with LPS + Nigericin group (positive control), * P < .05. D and E, The expression level of the pyroptosis‐related protein. The expression of GSDMD was relatively low in the control group, p30‐GSDMD was rarely expressed, and the level of cleaved caspase‐1 was also low. After Aβ1‐42 intervention, the expression of GSDMD, the upstream protein NLRP3 and p30‐GSDMD was significantly up‐regulated, while the expression of caspase‐11 was not obvious, indicating that Aβ1‐42 stimulated the expression of caspase‐1 to cleave GSDMD to cause pyroptosis. Comparison between groups, * P < .05

Article Snippet: To determine whether Aβ 1‐42 induced pyroptosis activating GSDMD oligomerization, the p30‐GSDMD oligomerization inhibitor, necrosulfonamide (NSA) (MCE, Shanghai, China) was used for intervention.

Techniques: Expressing, Cell Culture, Control, Permeability, Comparison, Positive Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: New mechanism of nerve injury in Alzheimer’s disease: β‐amyloid‐induced neuronal pyroptosis

doi: 10.1111/jcmm.15439

Figure Lengend Snippet: caspase‐1 or GSDMD silencing inhibited Aβ 1‐42 ‐induced pyroptosis in MCNs (n = 3). A, Results on the relative uptake rate of PI. The relative uptake rate of PI was significantly increased in Aβ 1‐42 intervention group, while siRNA‐caspase‐1 and siRNA‐GSDMD could significantly inhibit the relative uptake rate of PI, indicating that inhibition of caspase‐1 or GSDMD suppressed the opening of cell membranes and decreased PI uptake, compared to Aβ 1‐42 , * P < .05. B, Results on LDH cytotoxicity: Aβ 1‐42 intervention up‐regulated LDH release in MCNs, causing cytotoxicity. The cytotoxicity levels were down‐regulated in siRNA‐caspase‐1 and siRNA‐GSDMD groups, compared between groups, * P < .05. C and D, The mRNA expression of GSDMD and caspase‐1: Aβ 1‐42 intervention up‐regulated the expression of GSDMD and caspase‐1, while siRNA intervention down‐regulated the mRNA expression of GSDMD and caspase‐1. Comparison between groups, * P < .05. E, Results of PI and Hoechst 33 258 staining in MCNs: Aβ 1‐42 intervention caused relatively more positive‐staining cells, while GSDMD and caspase‐1 silencing resulted in significantly decreased number of positive cells, indicating that inhibition of caspase‐1 or GSDMD could decrease the opening level of the membrane pores. F, Results of GSDMD IF staining: After Aβ 1‐42 intervention, the GDXMD IF staining was relatively strong, indicating the high expression of GSDMD, while the levels of GSDMD were significantly decreased in siRNA‐caspase‐1 and siRNA‐GSDMD groups, suggesting that inhibition of caspase‐1 or GSDMD decreased the expression of GSDMD

Article Snippet: To determine whether Aβ 1‐42 induced pyroptosis activating GSDMD oligomerization, the p30‐GSDMD oligomerization inhibitor, necrosulfonamide (NSA) (MCE, Shanghai, China) was used for intervention.

Techniques: Inhibition, Expressing, Comparison, Staining, Membrane

Journal: Journal of Cellular and Molecular Medicine

Article Title: New mechanism of nerve injury in Alzheimer’s disease: β‐amyloid‐induced neuronal pyroptosis

doi: 10.1111/jcmm.15439

Figure Lengend Snippet: Effects of caspase‐1 or GSDMD silencing on Aβ 1‐42 ‐induced inflammatory factor release and expression of pyroptosis‐related protein in MCNs (n = 3). A‐C, The expression levels of inflammatory factors, including IL‐6, IL‐1β and TNF‐α in the culture medium. The expression of inflammatory factors was increased over time in the Aβ 1‐42 group, indicating increase cell permeability and the release of inflammatory factors. However, the levels of inflammatory factor were significantly down‐regulated in siRNA‐caspase‐1 and siRNA‐GSDMD groups, compared with Aβ1‐42 group, * P < .05. D and E, The expression of pyroptosis‐related proteins. The expression levels of GSDMD and p30‐GSDMD were higher in the Aβ 1‐42 group. After caspase‐1 inhibition, the level of p30‐GSDMD was down‐regulated, while the expression of GSDMD and NLRP3 was not significantly changed. After suppression of GSDMD, the levels of GSDMD and p30‐GSDMD were lower, but did not affect the expression of upstream cleaved proteins caspase‐1 and NLRP3. Comparison between groups, * P < .05

Article Snippet: To determine whether Aβ 1‐42 induced pyroptosis activating GSDMD oligomerization, the p30‐GSDMD oligomerization inhibitor, necrosulfonamide (NSA) (MCE, Shanghai, China) was used for intervention.

Techniques: Expressing, Permeability, Inhibition, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: New mechanism of nerve injury in Alzheimer’s disease: β‐amyloid‐induced neuronal pyroptosis

doi: 10.1111/jcmm.15439

Figure Lengend Snippet: p30‐GSDMD oligomerization inhibitor NSA inhibited Aβ 1‐42 ‐induced pyroptosis in MCNs (n = 3). A, Results of the relative uptake rate of PI: Aβ 1‐42 treatment could induce increased relative uptake rate of PI in MCNs, and NSA intervention could inhibit the opening of cell membrane and decrease PI uptake, compared with Aβ 1‐42 , * P < .05. B, Results of LDH cytotoxicity: Aβ 1‐42 treatment up‐regulated the release of LDH in MCNs, causing cytotoxicity. After NSA intervention, the cytotoxicity level was down‐regulated. Comparison between groups, * P < .05. C, Results of PI and Hoechst 33 258 staining: Aβ 1‐42 intervention increased the number of positive‐staining cells, while NSA intervention significantly decreased the number of positive‐staining cells, and NSA could attenuated the opening degree of membrane pores. D, Results of GSDMD IF staining: Aβ 1‐42 intervention caused relatively stronger IF staining of GDXMD, indicating the relatively higher expression of GSDMD; however, the level of GSDMD was not significantly changed after NSA intervention, suggesting that NSA did not affect the expression of GSDMD, only inhibited oligomerization of p30‐GSDMD

Article Snippet: To determine whether Aβ 1‐42 induced pyroptosis activating GSDMD oligomerization, the p30‐GSDMD oligomerization inhibitor, necrosulfonamide (NSA) (MCE, Shanghai, China) was used for intervention.

Techniques: Membrane, Comparison, Staining, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: New mechanism of nerve injury in Alzheimer’s disease: β‐amyloid‐induced neuronal pyroptosis

doi: 10.1111/jcmm.15439

Figure Lengend Snippet: p30‐GSDMD oligomerization inhibitor NSA inhibited Aβ 1‐42 ‐induced inflammatory factor release and expression of pyroptotic protein (n = 3). A‐C, The expression levels of inflammatory factors, including IL‐6, IL‐1β and TNF‐α in cell culture medium. The expression of these inflammatory factors was increased over time in the Aβ 1‐42 group, indicating the increased cell permeability and the release of inflammatory factors. And the levels of inflammatory factors were significantly down‐regulated after NSA intervention, compared with Aβ 1‐42 group, * P < .05. D and E, The expression level of pyroptosis‐related proteins. The expression levels of GSDMD and p30‐GSDMD (executive protein of pyroptosis) were relatively higher in the Aβ 1‐42 group; however, the levels of GSDMD and p30‐GSDMD were not significantly changed after NSA intervention. Meanwhile, the expression of caspase‐1 (cleavage protein) or upstream NLRP3 was not significantly changed. Comparison between groups, ns P > 0.05. These results indicated that NSA did not affect the expression of pyroptosis signalling protein nor did it affect the cleavage, but affected the oligomerization of the effector protein p30‐GSDMD and suppressed the oligomerization of p30‐GSDMD to open the membrane pore

Article Snippet: To determine whether Aβ 1‐42 induced pyroptosis activating GSDMD oligomerization, the p30‐GSDMD oligomerization inhibitor, necrosulfonamide (NSA) (MCE, Shanghai, China) was used for intervention.

Techniques: Expressing, Cell Culture, Permeability, Comparison, Membrane

Journal: ENERGY & ENVIRONMENTAL MATERIALS

Article Title: Reconstruction of Postinfarcted Cardiac Functions Through Injection of Tanshinone IIA@ Reactive Oxygen Species‐Sensitive Microspheres Encapsulated in a Thermoreversible Hydrogel

doi: 10.1002/eem2.12555

Figure Lengend Snippet: Figure 6. Cell pyroptosis and inhibition of inflammation. a) Immunofluorescence staining of the pyroptosis marker proteins N-GSDMD and GSDME in heart sections. b) Western blot of pyroptosis marker proteins and inflammatory factors. c) Immunohistochemical staining of inflammatory factors in heart sections. d) ELISA for quantitative analysis of inflammatory factors. n = 10. *p < 0.05, **p < 0.01, ***p < 0.001. 1) Sham group, 2) MI group, 3) Gel-MS group, 4) Gel-TIIA, and 5) Gel-MS/TIIA.

Article Snippet: Primary antibodies against NLRP-3, IL-1b, IL18, TNF-a, GSDMD, and GSDME were acquired from Cell Signaling Technology (Danvers, MA).

Techniques: Inhibition, Staining, Marker, Western Blot, Immunohistochemical staining, Enzyme-linked Immunosorbent Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Curcumin Ameliorates White Matter Injury after Ischemic Stroke by Inhibiting Microglia/Macrophage Pyroptosis through NF- κ B Suppression and NLRP3 Inflammasome Inhibition

doi: 10.1155/2021/1552127

Figure Lengend Snippet: Curcumin treatment reduces stroke-mediated NLRP3 inflammasome activation and pyroptosis in the ipsilateral peri-infarct regions 21 days after cerebral ischemia. (a, b) Representative western blot of NLRP3 and pyroptosis-related proteins in mice. Quantification of western blot data of NLRP3 (c), pro-caspase-1 (d), cleaved caspase-1 (e), GSDMD-FL (f), GSDMD-N (g), pro-IL-1 β (h), and IL-1 β (i). All the values are the mean ± SEM. ∗∗∗ p < 0.001. n = 8 mice per group, 1 band/mouse. One-way ANOVA followed by Bonferroni post hoc test. GSDMD-FL denotes GSDMD-full length. GSDMD-N indicates GSDMD-N-terminal.

Article Snippet: After blocking with 10% normal goat serum (ZSGB-BIO, China), the sections were incubated with a primary antibody mixture containing rabbit anti-Iba-1 antibody (1 : 500, ab178846, Abcam), mouse anti-GSDMD antibody (1 : 50, sc-393581, Santa Cruz)/mouse anti-caspase-1 antibody (1 : 50, sc-392736, Santa Cruz) for 36 hours at 4°C, and then incubated with a cocktail of solution mixture of mouse anti-rabbit IgG-CFL 594 (1 : 100, sc-516250, Santa Cruz) and mouse-IgG κ BP-CFL 488 (1 : 50, sc-516176, Santa Cruz) for 4 hours at room temperature.

Techniques: Activation Assay, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Curcumin Ameliorates White Matter Injury after Ischemic Stroke by Inhibiting Microglia/Macrophage Pyroptosis through NF- κ B Suppression and NLRP3 Inflammasome Inhibition

doi: 10.1155/2021/1552127

Figure Lengend Snippet: Curcumin inhibits microglial pyroptosis in lipopolysaccharide- (LPS-) and ATP-stimulated primary microglial cells. Primary microglia were extracted from the cortex of newborn C57BL/6J mice. Microglia were treated with vehicle or curcumin (12.5 μ M) in the presence or absence of LPS (100 ng/mL) for 24 hours followed by ATP (1 mM) stimulation for further 3 hours. (a, b) Representative western blot of pyroptosis-related proteins. Quantitative analysis of western blot data for NLRP3 (c), pro-caspase-1 (d), cleaved caspase-1 (e), GSDMD-FL (f), GSDMD-N (g), pro-IL-1 β (h), IL-18 (i), pro-IL-18 (j), and IL-18 (k). All the data are means ± SEM. n = 3 per group. Samples were collected from three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, one-way ANOVA followed by Bonferroni post hoc test. The mRNA expression of NLRP3 (l), caspase-1 (m), IL-18 (n), and IL-1 β (o) was examined by real-time PCR. All the data are means ± SEM. n = 3 per group. Samples were collected from three independent experiments. ∗∗ p < 0.01 and ∗∗∗ p < 0.001, one-way ANOVA followed by Bonferroni post hoc test.

Article Snippet: After blocking with 10% normal goat serum (ZSGB-BIO, China), the sections were incubated with a primary antibody mixture containing rabbit anti-Iba-1 antibody (1 : 500, ab178846, Abcam), mouse anti-GSDMD antibody (1 : 50, sc-393581, Santa Cruz)/mouse anti-caspase-1 antibody (1 : 50, sc-392736, Santa Cruz) for 36 hours at 4°C, and then incubated with a cocktail of solution mixture of mouse anti-rabbit IgG-CFL 594 (1 : 100, sc-516250, Santa Cruz) and mouse-IgG κ BP-CFL 488 (1 : 50, sc-516176, Santa Cruz) for 4 hours at room temperature.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Curcumin Ameliorates White Matter Injury after Ischemic Stroke by Inhibiting Microglia/Macrophage Pyroptosis through NF- κ B Suppression and NLRP3 Inflammasome Inhibition

doi: 10.1155/2021/1552127

Figure Lengend Snippet: Curcumin and NLRP3 inflammasome inhibition attenuate stroke-induced microglial pyroptosis in vivo and in vitro . In vivo , custom-made AAV vectors carrying shRNA targeting NLRP3 (NLRP3-shRNA) were used to silence the expression of NLRP3 for 14 days, followed by the operation of MCAO. Protein samples were extracted from the ipsilateral peri-infarct areas 21 days after stroke. In vitro , NLRP3 was knocked down with siRNA in primary microglia. Representative western blot of pyroptosis-associated proteins in vivo (a, b) and in vitro (j, k). Quantitative analysis of western blot data for NLRP3 (c, l), pro-caspase-1 (d, m), cleaved caspase-1 (e, n), GSDMD-FL (f, o), GSDMD-N (g, p), pro-IL-1 β (h, q), IL-1 β (i, r), pro-IL-18 (s), and IL-18 (t). Values are the mean ± SEM. In vivo , n = 5 mice per group, 1 band/mouse. In vitro , samples were collected from three independent experiments. n = 3 per group. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, one-way ANOVA followed by Bonferroni post hoc test.

Article Snippet: After blocking with 10% normal goat serum (ZSGB-BIO, China), the sections were incubated with a primary antibody mixture containing rabbit anti-Iba-1 antibody (1 : 500, ab178846, Abcam), mouse anti-GSDMD antibody (1 : 50, sc-393581, Santa Cruz)/mouse anti-caspase-1 antibody (1 : 50, sc-392736, Santa Cruz) for 36 hours at 4°C, and then incubated with a cocktail of solution mixture of mouse anti-rabbit IgG-CFL 594 (1 : 100, sc-516250, Santa Cruz) and mouse-IgG κ BP-CFL 488 (1 : 50, sc-516176, Santa Cruz) for 4 hours at room temperature.

Techniques: Inhibition, In Vivo, In Vitro, shRNA, Expressing, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Curcumin Ameliorates White Matter Injury after Ischemic Stroke by Inhibiting Microglia/Macrophage Pyroptosis through NF- κ B Suppression and NLRP3 Inflammasome Inhibition

doi: 10.1155/2021/1552127

Figure Lengend Snippet: Schematic diagram illustrating the inhibitory effect of curcumin on microglial pyroptosis and proinflammatory responses by NF- κ B signaling suppression and NLRP3 inhibition after stroke. Ischemic stroke triggers the activation of NF- κ B with the translocation of p50 and p65 into the nucleus in microglia, facilitating the transcription of target genes, such as GSDMD, NLRP3, IL-1 β , and IL-18. The oligomerization of NLRP3 with the ASC and pro-caspase-1 generates NLRP3 inflammasome, which activates caspase-1. Activated caspase-1 subsequently cleaves the GSDMD, pro-IL-1 β , and pro-IL-18 into GSDMD-N, IL-1 β , and IL-18, respectively. GSDMD-N translocates to the plasma membrane and eventually forms membrane pores, leading to the release of IL-1 β and IL-18. Thus, the microglial pyroptosis-mediated proinflammatory responses aggravate stroke-induced white matter damage. Curcumin dramatically suppresses NF- κ B signaling, inhibiting stroke-triggered NLRP3 upregulation and activation and microglial pyroptosis. Consequently, brain inflammation is significantly decreased, reversing white matter damage and function deficits.

Article Snippet: After blocking with 10% normal goat serum (ZSGB-BIO, China), the sections were incubated with a primary antibody mixture containing rabbit anti-Iba-1 antibody (1 : 500, ab178846, Abcam), mouse anti-GSDMD antibody (1 : 50, sc-393581, Santa Cruz)/mouse anti-caspase-1 antibody (1 : 50, sc-392736, Santa Cruz) for 36 hours at 4°C, and then incubated with a cocktail of solution mixture of mouse anti-rabbit IgG-CFL 594 (1 : 100, sc-516250, Santa Cruz) and mouse-IgG κ BP-CFL 488 (1 : 50, sc-516176, Santa Cruz) for 4 hours at room temperature.

Techniques: Inhibition, Activation Assay, Translocation Assay