Journal: Journal of Inflammation Research

Article Title: Electroacupuncture Inhibits the Early Neuroinflammatory Cascade Triggered by TLR2 in the Prodromal Period of PD

doi: 10.2147/JIR.S585729

Figure Lengend Snippet: EA can inhibit the activation of microglia in the pPD stage mediated by TLR2. ( A ) Western blot detection of TLR2 protein expression in the substantia nigra. ( B ) Western blot detection of MyD88 protein expression in the substantia nigra. ( C ) Western blot detection of p-NF-κB-p65 expression in the substantia nigra. ( D ) Western blot detection of NLRP3 protein expression in the substantia nigra. ( E ) Western blot detection of Caspase-1 protein expression in the substantia nigra. ( F ) Western blot detection of GSDMD protein expression in the substantia nigra. ( G ) Western blot detection of IL-1β protein expression in the substantia nigra. (n=6; compared with the control group, *** P <0.001; compared with the model group, ### P <0.001, ## P <0.01, # P <0.05).

Article Snippet: Subsequently, the primary antibodies—TLR2 (#bs-10472R, Beijing Bioss Biotechnology Co., Ltd., 1:200) or GSDMD (#bs-14287R, Beijing Bioss Biotechnology Co., Ltd., 1:200)—were applied, and the sections were incubated at 4°C overnight.

Techniques: Activation Assay, Western Blot, Expressing, Control

Journal: Cell reports

Article Title: Pyroptosis of pulmonary fibroblasts and macrophages through NLRC4 inflammasome leads to acute respiratory failure.

doi: 10.1016/j.celrep.2025.115479

Figure Lengend Snippet: Figure 5. Caspase-8/GSDME pathway in macrophage contributed to acute lung injury induced by high-dose flagellin (A and B) WT mice, Casp1/, NAIP/, or Nlrc4/ mice were injected with 10 mg LFn-flagellin/PA. Kaplan-Meier survival plots (A) and arterial oxygen saturation were monitored (B). (C) Casp1/ mice received 10 mg LFn-flagellin/PA. The lung Kf,c was determined. (D) WT mice or TLR5/-deficient mice received 10 mg LFn-flagellin/PA. Arterial oxygen saturation was monitored. (legend continued on next page)

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Critical commercial assays Thromboplastin-D Pacific Hemostasis Cat#100357 TAT ELISA kit Abcam Cat#ab137994 CytoTox 96 Non-Radioactive Cytotoxicity Assay Promega Cat#G1780 Plastic cuvette CHRONO-LOG Cat#367 IL-6 ELISA kit ThermoFisher Scientific Cat#88-7064-88 IL-1b ELISA kit ThermoFisher Scientific Cat#88-7013A-88 TNF-a ELISA kit ThermoFisher Scientific Cat#88-7324-88 Deposited data Raw data for single-cell RNA-seq This paper GEO:GSE269196 Experimental models: Cell lines Mouse Primary Bone Marrow Derived Macrophages This paper N/A Mouse Lung Epithelial Cells This paper N/A Mouse Lung Endothelial Cells This paper N/A Mouse Lung Fibroblast Cells This paper N/A Mouse Primary Alveolar Epithelial Cells Cell Biologics Cat#C57-6053 Mouse Primary Lung Microvascular Endothelial Cells Cell Biologics Cat#C57-6011 Experimental models: Organisms/strains C57BL/6J The Jackson Laboratory JAX:000664; RRID:IMSR_JAX:000664 Casp1 / Genentech Inc; Nobuhiko Kayagaki; Kayagaki et al.47 N/A Gsdmd / National Institute of Genetics, Japan; Toshihiko Shiroishi; Wu et al.21 N/A Gsdmdfl/fl National Institute of Genetics, Japan; Toshihiko Shiroishi; Wu et al.21 N/A NAIP / The Jackson Laboratory JAX:032660; RRID:IMSR_JAX:032660 Gsdme / The Jackson Laboratory JAX:032411; RRID:IMSR_JAX:032411 Tlr5 / The Jackson Laboratory JAX:008377; RRID:IMSR_JAX:008377 B6.Cg-Tg(Col1a2-cre/ERT,-ALPP) 7Cpd/2J The Jackson Laboratory JAX:029567; RRID:IMSR_JAX:029567 Casp8 / /Ripk3 / Genentech Inc; Nobuhiko Kayagaki; Lee et al.45 N/A Nlrc4 / Genentech Inc; Lee et al.45 N/A Software and algorithms FlowJo v10.07 FLOWJO https://www.flowjo.com/learn/flowjo- university/flowjo/getting-started-in-flowjo/ 131 GraphPad Prism 6 GraphPad https://www.graphpad.com/scientific- software/prism/ R Studio v. 4.3.6 RStudio https://cran.r-project.org/ (Continued on next page) 16 Cell Reports 44, 115479, April 22, 2025

Techniques: Injection

Journal: eLife

Article Title: A VgrG2b fragment cleaved by caspase-11/4 promotes Pseudomonas aeruginosa infection through suppressing the NLRP3 inflammasome

doi: 10.7554/elife.99939

Figure Lengend Snippet: Figure 2. P. aeruginosa VgrG2b is cleaved by caspase-11. (A) Screening strategy for identifying caspase-11 substrates in P. aeruginosa. Briefly, P. aeruginosa cDNAs were cloned into GFP vectors containing a mitochondrial localization signal. RFP vectors with a mitochondrial localization signal were used as transfection controls. HEK293T cells were co-transfected with these vectors and caspase-11. Cells losing GFP signals on mitochondria were sequenced. Plasmids encoding VgrG2b and mouse caspase-11 p22/p10 (B) or human caspase-4 p22/p10 (C) were co-transfected into HEK293T cells for 24 hr, followed by immunoblotting with antibodies against the indicated proteins. (D) Wild-type BMDM cells were primed overnight with 1 μg/ml Pam3CSK4 and incubated with ΔRetS PAO1 or VgrG2b-Myc knockin ΔRetS PAO1 at a multiplicity of infection (MOI) of 30 and 20 μg/ml outer membrane vesicles (OMVs) for 2 hr. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were lysed and immunoblotted as indicated 16 hr post infection. (E) Wild-type BMDM cells were primed overnight with 1 μg/ml Pam3CSK4 and incubated with VgrG2b-Myc knockin ΔRetS PAO1 at an MOI of 30 and 20 μg/ml OMVs for 2 hr. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were fixed and stained with antibodies against Myc or caspase-11 16 hr post infection. Nucleus was stained with DAPI. Scale bar, 5 μm. (F) Casp11+/+ and Casp11–/– BMDM cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml lipopolysaccharide (LPS) using DOTAP with or without the incubation of VgrG2b-Myc knockin ΔRetS PAO1 at an MOI of 30 for 2 hr. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were lysed and immunoblotted 16 hr post infection. (G) Casp11–/– BMDM cells were infected with lentiviruses encoding a control vector of caspase-11. Cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP with or without

Article Snippet: Casp11–/– (S- KO- 01332), Gsdmd–/– (S- KO- 12963), and Aim2–/– (S- KO- 09889) mice were purchased from Cyagen Biosciences (Jiangsu, China).

Techniques: Clone Assay, Transfection, Western Blot, Incubation, Knock-In, Infection, Membrane, Staining, Control, Plasmid Preparation

Journal: eLife

Article Title: A VgrG2b fragment cleaved by caspase-11/4 promotes Pseudomonas aeruginosa infection through suppressing the NLRP3 inflammasome

doi: 10.7554/elife.99939

Figure Lengend Snippet: Figure 4. VgrG2b C-terminus interacts with NLRP3. (A) Yeast two-hybrid screening was performed using VgrG2b C-terminus as bait and a mouse bone marrow cDNA library was screened. The interaction strength between bait and preys were visualized by X-α-gal assays. Plasmids encoding VgrG2b C-terminus (B) or full-length (C) and NLRP3 were co-transfected into HEK293T cells for 24 hr, followed by immunoprecipitation with a control IgG or antibody against Myc. Precipitates were immunoblotted as indicated. (D) VgrG2b C-terminus proteins were introduced into BMDM cells with the help of cell-penetrating peptides (CPP) for 6 hr. Cells were lysed and immunoprecipitated with a control IgG or antibody against Myc. Precipitates were immunoblotted as indicated. (E) BMDM cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml lipopolysaccharide (LPS) using DOTAP with the incubation of VgrG2b knockin (KI) ΔRetS PAO1 at a multiplicity of infection (MOI) of 30 for 2 hr. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were lysed and immunoprecipitated with a control IgG or antibody against NLRP3 16 hr post infection. Precipitates were immunoblotted as indicated. (F) BMDM cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ml LPS using DOTAP with the incubation of VgrG2b knockin (KI) ΔRetS PAO1 at an MOI of 30 for 2 hr. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were fixed and stained with antibodies against Myc and NLRP3 16 hr post infection. Nucleus was stained with DAPI. Scale bar, 5 μm. (G) Scheme for NLRP3 truncations. (H, I) Plasmids encoding VgrG2b C-terminus and NLRP3 truncations were co-transfected into HEK293T cells for 24 hr, followed by immunoprecipitation with a control IgG or antibody against FLAG. Precipitates were immunoblotted as indicated. (J) Casp11+/+ and Casp11–/– BMDM cells were primed overnight with 1 μg/ml Pam3CSK4, followed by transfection of 2 μg/ ml LPS using DOTAP with the incubation of VgrG2b-Myc knockin (KI) ΔRetS PAO1 at an MOI of 30 for 2 hr. Cells were then supplemented with fresh medium containing 100 μg/ml Gentamycin. Cells were lysed and immunoprecipitated with a control IgG or antibody against NLRP3 16 hr post infection. Precipitates were immunoblotted as indicated. Data were shown as means ± SD. Experiments were repeated three times with similar results.

Article Snippet: Casp11–/– (S- KO- 01332), Gsdmd–/– (S- KO- 12963), and Aim2–/– (S- KO- 09889) mice were purchased from Cyagen Biosciences (Jiangsu, China).

Techniques: Two Hybrid Screening, cDNA Library Assay, Transfection, Immunoprecipitation, Control, Incubation, Knock-In, Infection, Staining

Journal: American Journal of Cancer Research

Article Title: Cistanche phenylethanoid glycosides induce apoptosis and pyroptosis in T-cell lymphoma

doi: 10.62347/GEZW9659

Figure Lengend Snippet: CPhGs induced pyroptosis. HH and Hut78 cells were treated with indicated concentrations of CPhGs. A. Western blotting of cleaved GSDMD and GSDME (upper) with quantitative analysis (lower). B. Changes in LDH levels. C. Western blotting analysis of cleaved caspase-4 and caspase-1 (left) and the quantitative analysis (right).

Article Snippet: GSDMD and GSDME knockdown was achieved using siRNA. siRNA oligos targeting GSDMD (#SR312602), GSDME (#SR301192), or non-specific siRNAs were purchased from OriGene (Rockville, MD).

Techniques: Western Blot

Journal: American Journal of Cancer Research

Article Title: Cistanche phenylethanoid glycosides induce apoptosis and pyroptosis in T-cell lymphoma

doi: 10.62347/GEZW9659

Figure Lengend Snippet: CPhGs activated non-canonical and alternative pyroptotic pathways but not the canonical pyroptotic pathway. Cells were pre-treated with 20 µM ZVAD-FMK or/and VX765 followed by indicated concentrations of CPhGs. A. Cell survival rates following treatment. B. Changes in LDH levels. C. Western blotting results for specified proteins. D. Cells transfected with specified siRNAs, and the effects of GSDMD/E knockdown on cell viability were assessed (left). Western blot showing silencing effects of siRNAs (right). White, gray, and black columns represent control, CPhGs, and CPhGs+GSDMD/E siRNAs, respectively. Data are represented as the mean ± SD. **P < 0.01 compared to CPhGs only.

Article Snippet: GSDMD and GSDME knockdown was achieved using siRNA. siRNA oligos targeting GSDMD (#SR312602), GSDME (#SR301192), or non-specific siRNAs were purchased from OriGene (Rockville, MD).

Techniques: Western Blot, Transfection, Knockdown, Control

Journal: American Journal of Cancer Research

Article Title: Cistanche phenylethanoid glycosides induce apoptosis and pyroptosis in T-cell lymphoma

doi: 10.62347/GEZW9659

Figure Lengend Snippet: The molecular mechanisms of the anti-tumor properties of CPhGs. A schematic figure illustrates that CPhGs: A. Induce apoptosis by activating both intrinsic and extrinsic apoptotic pathways; B. Up-regulate pro-apoptotic factors PTEN and BAX, increase tumor-suppressor P53 by inhibiting SIRT2/MDM2 signaling, and suppress anti-apoptotic PI3K/AKT signaling; C. Induce pyroptosis by activating both caspase-4 (non-canonical pyroptosis) and caspase-3 (alternative pyroptosis) pathways; D. Inhibit proliferation by down-regulating NLRP3 and downstream IL-1β. Red lines denote inhibitory effects, and ‘x’ indicates blockade of functions. Italicized text in red shows three pyroptosis pathways. (+) and (-) denote activation and non-activation of the pathways, respectively. The figure is divided into two parts by a dotted line in the middle. The left part outlines the key components of apoptosis and their relationships. Both caspase-8 and -9 are activated to cleave the executor caspase-3, resulting in apoptosis and possibly an apoptosis-pyroptosis switch. The right part shows the mechanism of pyroptosis: GSDMD and GSDME are pyroptosis executors that form pores in the cell membrane, leading to K+ efflux, cell swelling, and death. VX765 is identified as a caspase 4 inhibitor, blocking non-canonical pyroptosis.

Article Snippet: GSDMD and GSDME knockdown was achieved using siRNA. siRNA oligos targeting GSDMD (#SR312602), GSDME (#SR301192), or non-specific siRNAs were purchased from OriGene (Rockville, MD).

Techniques: Activation Assay, Membrane, Blocking Assay

Journal: International journal of molecular sciences

Article Title: Implication of lncRNA MSTRG.81401 in Hippocampal Pyroptosis Induced by P2X7 Receptor in Type 2 Diabetic Rats with Neuropathic Pain Combined with Depression.

doi: 10.3390/ijms25021186

Figure Lengend Snippet: Figure 5. Expression of inflammasome/pyroptosis pathway proteins (A–G) and inflammatory factors (H–M) in hippocampus was examined by Western blotting. The bar graphs show the statistical data of relative expression levels of targeted molecules after being normalized by β-actin. NLRP3, GSDMD and ASC (A–D); procaspase-1 and cleaved-caspase-1 (E–G); TNF-α (H,I), IL-1β (J,K) and IL-18 (L,M). Values are mean ± SEM, n = 6; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Ctrl group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. DNP + MDD group.

Article Snippet: The primary antibodies used and their sources are as follows: P2X7 (1:800, APR-008, Alomone, Jerusalem, Israel), NLRP3 (1:800, IMG-6668A, Novus Biologicals, Centennial, CO, USA), GSDMD (1:800, NBP2-33422, Novus Biologicals, CO, USA), ASC (1:500, DF6304, Affinity Biosciences, Cincinnati, OH, USA), pro-caspase-1 (1:1000, AB179515, Abcam, Cambridge, UK), cleaved-caspase-1 (1:1000, AF4022, Affinity Biosciences, OH, USA), IL-1β (1:500, BA14789, Boster, Pleasanton, CA, USA), TNF-α (1:500, BA0131, Boster, CA, USA), IL-18 (1:500, DF6252, Affinity Biosciences, OH, USA), and β-actin (1:1000, Zhong Shan-Gold Bridge, Beijing, China).

Techniques: Expressing, Western Blot

Journal: Journal of healthcare engineering

Article Title: Reversing Postcardiopulmonary Bypass Associated Cognitive Dysfunction Using k -Opioid Receptor Agonists to Regulate Microglial Polarization via the NLRP3/Caspase-1 Pathway.

doi: 10.1155/2021/3048383

Figure Lengend Snippet: Figure 4: KORs inhibit cell pyroptosis through mediating cerebral pyrophosphorylation in CPB rats. (a) Expression levels of GSDMD and cleaved GSDMD-N-terminal in rats’ brain tissue samples were quantified with western blotting. (b) Counterstaining of GSDMD and TUNEL (scale bar 50 μm) was carried out for evaluation of pyrophoric and apoptotic rate, respectively. Relative pyroptotic and apoptotic rates were represented in the form of a bar chart. All data in charts were represented as mean ± standard deviation; “∗” was labeled when the comparative significance between groups (p value) was less than 0.05.

Article Snippet: GSDMD antibody (NBP2-80427, NOVUS, USA) staining was used to detect cellular pyroptosis, as GSDMD is the pyroptotic factor [29].

Techniques: Expressing, Western Blot, TUNEL Assay, Standard Deviation, Labeling