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

Journal: Brazilian Journal of Medical and Biological Research

Article Title: 25-hydroxycholesterol aggravates oxygen-glucose deprivation/reoxygenation-induced pyroptosis through promoting activation of NLRP3 inflammasome in H9C2 cardiomyocytes

doi: 10.1590/1414-431X2024e13299

Figure Lengend Snippet: Oxygen-glucose deprivation/reoxygenation (OGD/R) treatment promoted pyroptosis, NLRP3 inflammasome activation, and 25-hydroxylase (CH25H) expression in H9C2 cells. The H9C2 cells were subjected to oxygen-glucose deprivation for 6 h and then subjected to normoxic conditions for 3, 6, or 12 h. A , CCK-8 assay was used to detect cell viability. B , Lactate dehydrogenase (LDH) cytotoxicity detection assay was used to detect the LDH release. C , Hoechst 33342 and propidium iodide (PI) were used to stain cells. The nuclei were stained blue by Hoechst 33342, and the pyroptotic cells were stained red by PI; scale bars=100 μm. D , The mRNA and ( E ) protein expressions of CH25H, NLRP3, Caspase-1, GSDMD, IL-1β, and GAPDH were detected by RT-qPCR and western blotting. Data are reported as means±SD (n=3). *P<0.05, **P<0.01, and ***P<0.001 vs the control group (one-way ANOVA with Tukey multiple comparison test).

Article Snippet: Membranes were blocked with tris-buffered saline with tween 20 (TBST) buffer containing 5% nonfat milk and then incubated with primary antibodies against GAPDH (1:1000, Abcam, UK), Caspase-1 (1:1000, Abcam), NLRP3 (1:1000, Proteintech, China), GSDMD (1:1000, Biorbyt, UK), IL-1β (1:1000, Beyotime, China), and CH25H (1:500, Santa Cruz, USA) overnight at 4°C.

Techniques: Activation Assay, Expressing, CCK-8 Assay, Detection Assay, Staining, Quantitative RT-PCR, Western Blot, Control, Comparison

Journal: Brazilian Journal of Medical and Biological Research

Article Title: 25-hydroxycholesterol aggravates oxygen-glucose deprivation/reoxygenation-induced pyroptosis through promoting activation of NLRP3 inflammasome in H9C2 cardiomyocytes

doi: 10.1590/1414-431X2024e13299

Figure Lengend Snippet: CH25H knockdown inhibited pyroptosis and NLRP3 inflammasome activation in oxygen-glucose deprivation/reoxygenation (OGD/R)-treated H9C2 cardiomyocytes. The H9C2 cells were transfected with si-NC (negative control) or si-CH25H and then subjected to oxygen-glucose deprivation for 6 h following reoxygenation for 12 h. A , CCK-8 assay was used to detect cell viability. B , Lactate dehydrogenase (LDH) cytotoxicity detection assay was used to detect LDH release. C , Hoechst 33342 and propidium iodide (PI) were used to stain cells; scale bars=100 μm. The nuclei were stained blue by Hoechst 33342, and the pyroptotic cells were stained red by PI. D , The mRNA and ( E ) protein expressions of CH25H, NLRP3, Caspase-1, GSDMD, IL-1β, and GAPDH were detected by RT-qPCR and western blotting. Data are reported as means±SD (n=3). ***P<0.001 vs control group, ## P<0.01 and ### P<0.001 vs OGD/R+si-NC group (one-way ANOVA with Tukey multiple comparison test).

Article Snippet: Membranes were blocked with tris-buffered saline with tween 20 (TBST) buffer containing 5% nonfat milk and then incubated with primary antibodies against GAPDH (1:1000, Abcam, UK), Caspase-1 (1:1000, Abcam), NLRP3 (1:1000, Proteintech, China), GSDMD (1:1000, Biorbyt, UK), IL-1β (1:1000, Beyotime, China), and CH25H (1:500, Santa Cruz, USA) overnight at 4°C.

Techniques: Knockdown, Activation Assay, Transfection, Negative Control, CCK-8 Assay, Detection Assay, Staining, Quantitative RT-PCR, Western Blot, Control, Comparison

Journal: Brazilian Journal of Medical and Biological Research

Article Title: 25-hydroxycholesterol aggravates oxygen-glucose deprivation/reoxygenation-induced pyroptosis through promoting activation of NLRP3 inflammasome in H9C2 cardiomyocytes

doi: 10.1590/1414-431X2024e13299

Figure Lengend Snippet: 25-HC promoted NLRP3 inflammasome activation in oxygen-glucose deprivation/reoxygenation (OGD/R)-stimulated H9C2 cardiomyocytes. The H9C2 cells were treated with 25-HC at different concentrations (0.4, 2, 10, or 50 μM) during reoxygenation. The si-CH25H-transfected H9C2 cells were treated with 25-HC (0.4 μM) during reoxygenation. A and D , Cell viability was detected by CCK-8 assay. B and E , Lactate dehydrogenase (LDH) release was detected by LDH cytotoxicity detection assay. C and F , The protein expressions of NLRP3, Caspase-1, GSDMD, IL-1β, and GAPDH were detected by western blotting. Data are reported as means±SD (n=3). ***P<0.001 vs control group, # P<0.05, ## P<0.01, and ### P<0.001 vs OGD/R group, && P<0.01 and &&& P<0.001 vs OGD/R+si-CH25H group (one-way ANOVA with Tukey multiple comparison test).

Article Snippet: Membranes were blocked with tris-buffered saline with tween 20 (TBST) buffer containing 5% nonfat milk and then incubated with primary antibodies against GAPDH (1:1000, Abcam, UK), Caspase-1 (1:1000, Abcam), NLRP3 (1:1000, Proteintech, China), GSDMD (1:1000, Biorbyt, UK), IL-1β (1:1000, Beyotime, China), and CH25H (1:500, Santa Cruz, USA) overnight at 4°C.

Techniques: Activation Assay, Transfection, CCK-8 Assay, Detection Assay, Western Blot, Control, Comparison

Journal: Brazilian Journal of Medical and Biological Research

Article Title: 25-hydroxycholesterol aggravates oxygen-glucose deprivation/reoxygenation-induced pyroptosis through promoting activation of NLRP3 inflammasome in H9C2 cardiomyocytes

doi: 10.1590/1414-431X2024e13299

Figure Lengend Snippet: 25-HC promoted oxygen-glucose deprivation/reoxygenation (OGD/R)-induced pyroptosis through activation of NLRP3 inflammasome in H9C2 cardiomyocytes. The H9C2cells were pretreated with NLRP3 inflammasome inhibitor BAY11-7082 (2 μM) 2 h before OGD/R treatment. Then, cells were treated with 25-hydroxycholesterol (0.4 μM) during reoxygenation. A , CCK-8 assay was used to detect cell viability. B , Lactate dehydrogenase (LDH) cytotoxicity detection assay was used to detect the LDH release. C , Hoechst 33342 and propidium iodide (PI) were used to stain cells; scale bars=100 μm. The nuclei were stained blue by Hoechst 33342, and the pyroptotic cells were stained red by PI. D , The protein expressions of NLRP3, Caspase-1, GSDMD, IL-1β, and GAPDH were detected by western blotting. Data are reported as means±SD (n=3). ***P<0.001 vs control group, ## P<0.01 and ### P<0.001 vs OGD/R group, && P<0.01 and &&& P<0.001 vs OGD/R+25-HC group (one-way ANOVA with Tukey multiple comparison test).

Article Snippet: Membranes were blocked with tris-buffered saline with tween 20 (TBST) buffer containing 5% nonfat milk and then incubated with primary antibodies against GAPDH (1:1000, Abcam, UK), Caspase-1 (1:1000, Abcam), NLRP3 (1:1000, Proteintech, China), GSDMD (1:1000, Biorbyt, UK), IL-1β (1:1000, Beyotime, China), and CH25H (1:500, Santa Cruz, USA) overnight at 4°C.

Techniques: Activation Assay, CCK-8 Assay, Detection Assay, Staining, Western Blot, Control, Comparison

Journal: Cell reports

Article Title: Gasdermin E permits interleukin-1 beta release in distinct sublytic and pyroptotic phases

doi: 10.1016/j.celrep.2021.108998

Figure Lengend Snippet: (A and B) PMA-differentiated (100 ng/mL, 16 h) wild-type, GSDMD −/− , and GSDMD −/− GSDME −/− THP-1 cells treated with nigericin (20 μM) assessed for propidium iodide uptake, cell lysis through supernatant lactate dehydrogenase activity assay (A), and cell morphology by microscopy (B). (C and D) Immunoblots of these cells treated with nigericin or S. typhimurium (MOI = 10). (E) Mature IL-1β ELISA for supernatants of wild-type and indicated gasdermin knockout differentiated THP-1 treated with nigericin (20 μM). Immunoblots are representative of at least five independent experiments. Scale bar, 50 μm. Graph bars represent mean ± standard error of biological replicates. Graph points represent pooled technical replicates per biological replicate. See also .

Article Snippet: Human-specific antibodies were GSDMD (HPA044487, Atlas Antibodies) and IL-1β (AF-201-NA, R&D Systems).

Techniques: Lysis, Activity Assay, Microscopy, Western Blot, Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: Cell reports

Article Title: Gasdermin E permits interleukin-1 beta release in distinct sublytic and pyroptotic phases

doi: 10.1016/j.celrep.2021.108998

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human-specific antibodies were GSDMD (HPA044487, Atlas Antibodies) and IL-1β (AF-201-NA, R&D Systems).

Techniques: Recombinant, Virus, Live Cell Imaging, CyQUANT Assay, LDH Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: Molecular Metabolism

Article Title: Maternal high-fat diet exaggerates diet-induced insulin resistance in adult offspring by enhancing inflammasome activation through noncanonical pathway of caspase-11

doi: 10.1016/j.molmet.2020.100988

Figure Lengend Snippet: Noncanonical inflammasome activation pathway of caspase-11 is augmented in eWAT of O-HFD . ( A and B ) Representative ex vivo images of eWAT, as well as quantitative measurement of radiant efficiency corresponding to caspase-1 activity. Values are the mean ± SEM for 4 O-ND and 5 O-HFD mice. O-ND, offspring of ND-fed dam; O-HFD, offspring of HFD-fed dam. ( C and D ) Representative western blot of GSDMD and caspase-11 in eWAT, as well as quantitative analysis of protein expression. Values are the mean ± SEM for 5 O-ND and 5 O-HFD mice. ∗ P < 0.05 and ∗∗ P < 0.01 versus O-ND. O-ND, offspring of ND-fed dam; O-HFD, offspring of HFD-fed dam; GSDMD-FL, full length of gasdermin D; GSDMD-NT, N-terminal portion of gasdermin D.

Article Snippet: Protein samples were subjected to SDS-PAGE and then transferred to membranes that were subsequently incubated with primary antibodies against GSDMD (STJ112203; St John's Laboratory, London, UK), caspase-11 (ab180673, EPR18628; Abcam, Cambridge, UK), caspase-1 (EPR16883, ab179515, Abcam), α-tubulin (T5168, B-5-1-2, Sigma–Aldrich, St. Louis, MO, USA), and β-actin (A2228, AC-74; Sigma–Aldrich).

Techniques: Activation Assay, Ex Vivo, Activity Assay, Western Blot, Expressing