Journal: Poultry Science

Article Title: Selenoprotein S ablation-mediated pyroptosis contributes to liver damage resulting from selenium deficiency in chickens

doi: 10.1016/j.psj.2025.105269

Figure Lengend Snippet: SELS knockdown induces pyroptosis. (A-B) Western blot and qRT-PCR shows protein and transcription levels of SELS in primary hepatocytes and LMH cells. (C) Immunofluorescence images and quantification of NLRP3 and GSDMD in primary hepatocytes. (D-E) Western blot shows protein levels of pyroptosis-related genes in primary hepatocytes and LMH cells. (F) QRT-PCR detects transcript levels of pyroptosis-related genes in primary hepatocytes and LMH cells. All data were biologically replicated ( n = 3). A significant difference " p < 0.05″ is indicated by "*", otherwise " p > 0.05″ is labeled "ns", when compared with control group using t-test. Bars labeled with different letters indicate significant differences screened by one-way ANOVA.

Article Snippet: After blocking with 5 % BSA for 30 min, primary antibodies against NLRP3 (ABclonal, A12694, 1:100) or GSDMD (ABclonal, A24476 , 1:200) were added overnight at 4 °C followed by incubation with HRP-conjugated secondary antibody (Servicebio, GB23303, 1:200) for 45 min. After treatment with 3,3′-diaminobenzidine solution (Servicebio, G1212), images were captured using a fluorescence microscope.

Techniques: Knockdown, Western Blot, Quantitative RT-PCR, Immunofluorescence, Labeling, Control

Journal: Poultry Science

Article Title: Selenoprotein S ablation-mediated pyroptosis contributes to liver damage resulting from selenium deficiency in chickens

doi: 10.1016/j.psj.2025.105269

Figure Lengend Snippet: NF-κB is the upstream pathway of pyroptosis. (A-B) Western blot and qRT-PCR shows protein and transcription levels of pyroptosis-related genes in primary hepatocytes. (C-D) Western blot and qRT-PCR displays protein and transcription levels of pyroptosis-related genes in LMH cells. (E-F) Immunofluorescence images and quantification of NLRP3 and GSDMD in primary hepatocytes. All data were biologically replicated ( n = 3). Bars labeled with different letters indicate significant differences screened by one-way ANOVA.

Article Snippet: After blocking with 5 % BSA for 30 min, primary antibodies against NLRP3 (ABclonal, A12694, 1:100) or GSDMD (ABclonal, A24476 , 1:200) were added overnight at 4 °C followed by incubation with HRP-conjugated secondary antibody (Servicebio, GB23303, 1:200) for 45 min. After treatment with 3,3′-diaminobenzidine solution (Servicebio, G1212), images were captured using a fluorescence microscope.

Techniques: Western Blot, Quantitative RT-PCR, Immunofluorescence, Labeling

Journal: Poultry Science

Article Title: Selenoprotein S ablation-mediated pyroptosis contributes to liver damage resulting from selenium deficiency in chickens

doi: 10.1016/j.psj.2025.105269

Figure Lengend Snippet: ROS regulates pyroptosis of hepatocytes. (A-B) Immunofluorescence images and quantification of NLRP3 and GSDMD in hepatocytes. (C-D) Western blot shows protein levels of pyroptosis-related genes in hepatocytes and LMH cells. (E-F) QRT-PCR detects transcription levels of pyroptosis-related genes in hepatocytes and LMH cells. (G-H) The mRNA expression of the NF-κB pathway in primary hepatocytes and LMH cells. All data were biologically replicated ( n = 3). Bars labeled with different letters indicate significant differences screened by one-way ANOVA.

Article Snippet: After blocking with 5 % BSA for 30 min, primary antibodies against NLRP3 (ABclonal, A12694, 1:100) or GSDMD (ABclonal, A24476 , 1:200) were added overnight at 4 °C followed by incubation with HRP-conjugated secondary antibody (Servicebio, GB23303, 1:200) for 45 min. After treatment with 3,3′-diaminobenzidine solution (Servicebio, G1212), images were captured using a fluorescence microscope.

Techniques: Immunofluorescence, Western Blot, Quantitative RT-PCR, Expressing, Labeling

Journal: European Journal of Medical Research

Article Title: High-fat diet activates pyroptosis of retinal pigment epithelial cells in aged TgAPPswePS1 transgenic mice

doi: 10.1186/s40001-025-02898-5

Figure Lengend Snippet: Immunofluorescence analysis of the pyroptosis-related proteins in RPE cells. Scale bar = 20 μm. A Representative images showing the expressions of NLRP3, caspase-1 and GSDMD in RPE cells. B Representative images showing the expressions of IL-1β and IL-18 in RPE cells. C Semi-quantitative analysis of fluorescence intensity for NLRP3, caspase-1, GSDMD, IL-1β and IL-18 expressions in RPE cells. ** p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significance

Article Snippet: After blocking with 5% nonfat dry milk, membranes were incubated overnight at 4 °C with primary antibodies against NLRP3 (1:1000, #15101), Pro-Caspase-1 (1:1000, #24232), Cleaved-Caspase-1 (1:1000, #89332), Cleaved-GSDMD (1:1000, #10137, Cell Signaling Technology, MA, USA), GSDMD (1:1000, ab219800) and β-actin (1:1000, ab8226, Abcam Biotechnology, UK).

Techniques: Immunofluorescence, Fluorescence

Journal: European Journal of Medical Research

Article Title: High-fat diet activates pyroptosis of retinal pigment epithelial cells in aged TgAPPswePS1 transgenic mice

doi: 10.1186/s40001-025-02898-5

Figure Lengend Snippet: Expression alternations of pyroptosis-related proteins in RPE cells. A NLRP3, caspase-1, GSDMD protein expressions in RPE were detected via western blotting. B IL-1β and IL-18 levels in RPE were tested via ELISA. * p < 0.05, **p < 0.01, ***p < 0.001, ns, no significance

Article Snippet: After blocking with 5% nonfat dry milk, membranes were incubated overnight at 4 °C with primary antibodies against NLRP3 (1:1000, #15101), Pro-Caspase-1 (1:1000, #24232), Cleaved-Caspase-1 (1:1000, #89332), Cleaved-GSDMD (1:1000, #10137, Cell Signaling Technology, MA, USA), GSDMD (1:1000, ab219800) and β-actin (1:1000, ab8226, Abcam Biotechnology, UK).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: MicroRNAs as regulators of NLRP3 inflammasome activation in herpes simplex virus type 2 infection

doi: 10.3389/fcimb.2025.1602965

Figure Lengend Snippet: HSV-2 infection triggers inflammatory responses: Graphical representation of the results of the qRT-PCR analysis showing relative mRNA expression levels of (A) NLRP3, (B) ASC, (C) CASP1, and (D) UL30 in THP-1 cells infected with HSV-2 (MOI = 1) at 4-, 8-, 16-, and 24-hpi, after normalization with the internal control, GAPDH. Mock-infected cells served as a negative control, while lipopolysaccharides (LPS) treatment was used as a positive control. (E) Immunoblot analysis demonstrating the post-infection kinetics of the protein expression levels of NLRP3, ASC, Cleaved CASP1, HSV-2 ICP8, and GAPDH in THP-1 cells at 4-, 8-, 16-, and 24-hpi compared to the mock-infected samples. (F) Comparison of the protein expression levels of NLRP3, ASC, Cleaved CASP1, HSV-2 ICP8, and GAPDH in mock-infected, HSV-2 infected (MOI=1) and uninfected, LPS-treated THP-1 cells at 24 hours. GAPDH was used as a loading control for normalization. Data represent the mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. The number of asterisks, as shown in each of the graphs, indicates the level of significance of the data (*p<0.05, **p<0.01, ***p<0.001).

Article Snippet: The membrane was blocked with 5% non-fat dried milk for 1 hour at room temperature and incubated with primary antibodies specific to NLRP3 (15101), ASC (13833), Cleaved CASP1 (p20 subunit - Asp297; 4199), IL-1β (12703), IL-18 (57058), Cleaved GSDMD (Asp275; 36425) procured from Cell Signaling Technology (Danvers, MA, USA), HSV-2 gB (57857; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and HSV-2 ICP8 (56992; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Infection, Quantitative RT-PCR, Expressing, Control, Negative Control, Positive Control, Western Blot, Comparison

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: MicroRNAs as regulators of NLRP3 inflammasome activation in herpes simplex virus type 2 infection

doi: 10.3389/fcimb.2025.1602965

Figure Lengend Snippet: Schematic representation of the key signaling pathways involved in HSV-2-induced inflammation and pyroptosis: 1. HSV-2 Entry: Herpes Simplex Virus Type 2 (HSV-2) enters the host cell, initiating inflammatory responses. 2. Inflammasome Assembly: Upon HSV-2 infection, the miRNAs, miR-141 and miR-211 that target NLRP3 and CASP, are downregulated- a mechanism that may be crucial in facilitating the assembly of the inflammasome complex containing NLRP3, ASC, and Pro-Caspase-1. 3. Activation of Caspase-1: The assembled inflammasome activates Caspase-1 by cleaving its precursor, pro-Caspase-1. Activated Caspase-1 facilitates downstream inflammatory responses. 4. Cytokine Maturation: Caspase-1 in its active form cleaves the pro-inflammatory cytokine precursor Pro-IL-1β into its active form, IL-1β, which is then secreted excessively to amplify the inflammatory response. 5. Pyroptosis and Pore Formation: Caspase-1 also converts Gasdermin-D (GSDM-D) by cleaving into its N-terminal active fragment (N-GSDM-D), forming membrane pores. This results in pyroptosis, a form of programmed cell death, and the release of excessive inflammatory cytokines. 6. Ectopic Expression of miRNAs: MicroRNAs hsa-miR-141 and hsa-miR-211 are potential therapeutic agents. Their ectopic expressions suppress the inflammasome assembly and activation by targeting the key components NLRP3 and Caspase-1, respectively, thus mitigating inflammatory responses and viral replication. 7. By restricting inflammasome activation , these miRNAs limit the severity of HSV-2 infection, demonstrating their potential as therapeutic agents to inhibit inflammation and control viral pathogenesis.

Article Snippet: The membrane was blocked with 5% non-fat dried milk for 1 hour at room temperature and incubated with primary antibodies specific to NLRP3 (15101), ASC (13833), Cleaved CASP1 (p20 subunit - Asp297; 4199), IL-1β (12703), IL-18 (57058), Cleaved GSDMD (Asp275; 36425) procured from Cell Signaling Technology (Danvers, MA, USA), HSV-2 gB (57857; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and HSV-2 ICP8 (56992; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Protein-Protein interactions, Virus, Infection, Activation Assay, Membrane, Expressing, Control