Journal: Respiratory Research

Article Title: HGF alleviates septic endothelial injury by inhibiting pyroptosis via the mTOR signalling pathway

doi: 10.1186/s12931-020-01480-3

Figure Lengend Snippet: HGF alleviated endothelial pyroptosis in vitro. EA.hy926 cells were stimulated with LPS (2.5 μg/mL) with or without Lipo2000 (2 μL/mL), followed by administration of HGF (25 ng/mL) immediately and 6 h later. a Bright-field image of treated EA.hy926 cells from randomly selected fields of view, in which morphological changes characteristic of pyroptosis were assessed; scale bar = 100 μm; b Relative paracellular permeability of single-layer EA.hy926 cells exposed to different treatments and analysed by FITC-dextran; c IL-1β in the supernatant of treated EA.hy926 cells was measured by ELISA; d LDH release assay was performed to measure pyroptosis of the treated EA.hy926 cells, n = 3, * P < 0.05, ** P < 0.01; e CASP-1, CASP-1-P10, GSDMD and GSDMD-N in the treated EA.hy926 cell homogenates were measured by Western blot; n = 3

Article Snippet: The mouse monoclonal antibodies against gasdermin D (GSDMD) (sc-393,581) and β-actin (sc-517,582) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: In Vitro, Permeability, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Western Blot

Journal: Respiratory Research

Article Title: HGF alleviates septic endothelial injury by inhibiting pyroptosis via the mTOR signalling pathway

doi: 10.1186/s12931-020-01480-3

Figure Lengend Snippet: HGF ameliorated endothelial cell pyroptosis by promoting mTOR signalling. a EA.hy926 cells were treated as described, and images were captured from randomly selected fields of view by bright field microscopy to assess the morphological changes characteristic of pyroptosis; scale bar = 50 μm; b Relative paracellular permeability of single-layer EA.hy926 cells exposed to different treatments was measured by FITC-dextran; c IL-1β in the supernatant of treated EA.hy926 cells was measured by ELISA; d LDH release assay was performed to measure pyroptosis of treated EA.hy926 cells; e CASP-1, CASP-1-P10, GSDMD and GSDMD-N in the treated EA.hy926 cell homogenates were measured by Western blot; n = 3, * P < 0.05 compared with the tLPS group; # P < 0.05 compared with the tLPS+HGF group; PHA: PHA-665752, RAPA: rapamycin

Article Snippet: The mouse monoclonal antibodies against gasdermin D (GSDMD) (sc-393,581) and β-actin (sc-517,582) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Microscopy, Permeability, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Western Blot

Journal: Lipids in Health and Disease

Article Title: 4-phenylbutyric acid attenuates hepatic ER stress, pyroptosis, and NAFLD in adult rat offspring exposed to a high-fat, high-fructose diet during perinatal and adulthood

doi: 10.1186/s12944-026-02882-w

Figure Lengend Snippet: Experimental design. N: Normal diet, HFF: High-fat, high-fructose diet, DMSO: Dimethyl sulfoxide, 4PBA: 4-phenylbutyric acid, AST: Aspartate aminotransferase, ALT: Alanine aminotransferase, FFA: Free fatty acids, LDL: Low‑density lipoprotein, HDL: High‑density lipoprotein, UA: Uric acid, GSDMD: Gasdermin D, BIP: Binding immunoglobulin protein, CHOP: C/EBP homologous protein, IDE: Insulin‑degrading enzyme, SOD: Superoxide dismutase, GSH: Glutathione (reduced form), MDA: Malondialdehyde, IL‑1β: Interleukin‑1 beta, TNF‑α: Tumor necrosis factor alpha, OGTT: Oral glucose tolerance test, ITT: Insulin tolerance test

Article Snippet: The separated proteins were blotted onto PVDF membranes (Merck Millipore, Darmstadt, Germany) at 100 V for 85 min and the membranes were blocked with 5% BSA (Merck, Darmstadt, Germany) in 0.1% Tween-20 for 90 min at ambient temperature (24 °C) and then incubated overnight at 4 °C with primary antibodies against GSDMD (Cat. No.: 39754 S Cell Signaling Technology, Danvers, MA, USA), BIP (Cat. No.: 3183 S, Cell Signaling Technology, Danvers, MA, USA), CHOP (Cat. No.: 5554 S, Cell Signaling Technology, Danvers, MA, USA) and β-actin (for BIP and CHOP, 1:4000, Cat. No.: 4970, Cell Signaling Technology, Danvers, MA, USA; for GSDMD, 1:2500, Cat. No: ab8227, Abcam, Cambridge, UK).

Techniques: Binding Assay

Journal: Lipids in Health and Disease

Article Title: 4-phenylbutyric acid attenuates hepatic ER stress, pyroptosis, and NAFLD in adult rat offspring exposed to a high-fat, high-fructose diet during perinatal and adulthood

doi: 10.1186/s12944-026-02882-w

Figure Lengend Snippet: The hepatic expression level of ( a , b ) BIP, a , c CHOP, and d , e GSDMD proteins across different dietary intervention groups. Data points and columns represent mean ± SEM ( n = 3 per group). The vertical dashed line separates the dietary intervention groups from the pharmacological intervention groups. Groups 1–4 (N-N, N-HFF, HFF-N, and HFF-HFF) were compared separately to evaluate the effect of dietary interventions (significance indicated by asterisks). Groups 1, 4, 5, and 6 (N-N, HFF-HFF, HFF-HFF-DMSO, and HFF-HFF-4PBA) were analyzed separately to evaluate the effect of 4-PBA treatment (significance indicated by hash symbols). Statistical significance between connected groups are as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, # P < 0.05, ### P < 0.001, #### P < 0.0001. N: Normal diet, HFF: High-fat, high-fructose diet, DMSO: Dimethyl sulfoxide, 4PBA: 4-phenylbutyric acid, BIP: Binding immunoglobulin protein, CHOP: C/EBP Homologous protein, GSDMD: Gasdermin D

Article Snippet: The separated proteins were blotted onto PVDF membranes (Merck Millipore, Darmstadt, Germany) at 100 V for 85 min and the membranes were blocked with 5% BSA (Merck, Darmstadt, Germany) in 0.1% Tween-20 for 90 min at ambient temperature (24 °C) and then incubated overnight at 4 °C with primary antibodies against GSDMD (Cat. No.: 39754 S Cell Signaling Technology, Danvers, MA, USA), BIP (Cat. No.: 3183 S, Cell Signaling Technology, Danvers, MA, USA), CHOP (Cat. No.: 5554 S, Cell Signaling Technology, Danvers, MA, USA) and β-actin (for BIP and CHOP, 1:4000, Cat. No.: 4970, Cell Signaling Technology, Danvers, MA, USA; for GSDMD, 1:2500, Cat. No: ab8227, Abcam, Cambridge, UK).

Techniques: Expressing, Binding Assay

Journal: Redox biology

Article Title: Sulfhydrated albumin transmits H 2 S signaling and ameliorates DOX-induced multiorgan injuries.

doi: 10.1016/j.redox.2025.103631

Figure Lengend Snippet: Fig. 6. -SSH-Alb inhibits inflammasome activation and pyroptosis in macrophages. (A–F) Effects of differentially modified albumin or increasing concentra tions of –SSH–Alb on LPS/ATP-induced inflammasome activation and pyroptosis. BMDMs were pretreated with 5 mg/ml of various albumin preparations or increasing concentrations of –SSH–Alb for 30 min, followed by stimulation with 10 μg/ml LPS for 5.5 h and 5 mM ATP for an additional 30 min. IL-1β release in the culture supernatant was measured by ELISA (A, F). Cellular lysates were analyzed for NLRP3 and GSDMD-NT by western blotting (B, E), and band intensities of (B) were quantified (C, D) (mean ± SE, n = 4; **p < 0.01 vs. NC; ##p < 0.01). (G–J) Effects of differentially modified albumin or increasing concentrations of –SSH–Alb on LPS/ATP-induced oxidative stress. Cells were treated as described above. Cellular lysates were analyzed for –SOH (sulfenic acid) modification and phosphorylated P38 (p-P38) by western blotting (G, J), and band intensities of (G) were quantified (H, I) (mean ± SE, n = 3–4; *p < 0.05, **p < 0.01 vs. NC; ##p < 0.01). (K–P) Effect of GSH on LPS/ATP-induced pyroptosis and oxidative stress. BMDMs were pretreated with 5 mM GSH or left untreated for 1 h, followed by stimulation with 10 μg/ml LPS for 5.5 h and 5 mM ATP for 30 min. Culture medium and cell lysates were collected and analyzed for IL-1β, NLRP3, GSDMD-NT, –SOH, and p-P38. Quantitative data are shown in (K, M − P) (mean ± SE, n = 3; *p < 0.05, **p < 0.01 vs. Ctrl; #p < 0.05, ##p < 0.01).

Article Snippet: HRPconjugated anti-rabbit or mouse IgG (cat no.7776S or 7074S), anti- β-actin (cat no.A5316), anti-GPX4 (cat no.52455), anti-GSDMD-NT(cat no.10137), anti-phospho-Ser157 VASP (cat no.3111), and anti-p-P38 antibodies (cat no.9662) were purchased from Cell Signaling, Inc. (Beverly, MA, USA).

Techniques: Activation Assay, Modification, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Redox biology

Article Title: Sulfhydrated albumin transmits H 2 S signaling and ameliorates DOX-induced multiorgan injuries.

doi: 10.1016/j.redox.2025.103631

Figure Lengend Snippet: Fig. 7. -SSH-Alb alleviates doxorubicin (DOX)-induced cardiotoxicity and intestinal injury. (A) Schematic diagram of the experimental design. For acute cardiotoxicity induction, mice received a single intraperitoneal injection of DOX (25 mg/kg) or normal saline (NS). –SSH–Alb (1.5 g/kg) was administered five times at 12–24 h intervals. Mice were euthanized 72 h after DOX treatment, and tissue samples were collected. (B) Effect of –SSH–Alb on DOX-induced mortality. Mice received a single intraperitoneal injection of DOX (25 mg/kg), and mortality was monitored for 7 days (mean ± SE, n = 6; *p < 0.05). (C–E) Effects of –SSH–Alb on DOX-induced cardiac ferroptosis. Heart tissue lysates were analyzed for ferroptosis markers (x-CT, GPX4) by western blotting (C), and band intensities were quantified (D, E) (mean ± SE, n = 6; **p < 0.01 vs. Ctrl; #p < 0.05, ##p < 0.01). (F, G) Effects of –SSH–Alb on DOX-induced colon shortening. Colon was pho tographed (F) and measured for its length (F), and data are presented in (G) (mean ± SE, n = 6; **p < 0.01 vs. Ctrl; ##p < 0.01). (H–K) Effects of –SSH–Alb on DOX- induced colon injury and barrier dysfunction. Colon tissue lysates were analyzed for injury markers (lipocalin-2, GSDMD-NT) and the adherens junction protein E- cadherin by western blotting (H), and band intensities were quantified (I–K) (mean ± SE, n = 6; *p < 0.05, **p < 0.01 vs. Ctrl; ##p < 0.01).

Article Snippet: HRPconjugated anti-rabbit or mouse IgG (cat no.7776S or 7074S), anti- β-actin (cat no.A5316), anti-GPX4 (cat no.52455), anti-GSDMD-NT(cat no.10137), anti-phospho-Ser157 VASP (cat no.3111), and anti-p-P38 antibodies (cat no.9662) were purchased from Cell Signaling, Inc. (Beverly, MA, USA).

Techniques: Injection, Saline, Western Blot

Journal: Science advances

Article Title: Isocyanic acid-mediated NLRP3 carbamoylation reduces NLRP3-NEK7 interaction and limits inflammasome activation.

doi: 10.1126/sciadv.adq4266

Figure Lengend Snippet: Fig. 1. LACC1 limits NLRP3 inflammasome activation. PMA-differentiated THP-1 cells and iBMDMs without or with LACC1/Lacc1 KO were stimulated with LPS (100 ng ml−1) for 8 hours (A) and then incubated with 10 μM Nig or 5 mM ATP for another 45 min (B to G). P values were calculated using one-way ANOVA (B to E). The data are presented as mean ± SD of three (E) or four (B to D) independent experiments. *P < 0.01, #P < 0.05. (A) LACC1 in the lysates of indicated cells was analyzed by immunob- lotting. (B to D) The release of IL-1β (B), IL-18 (C), and LDH (D) in culture supernatants was determined by ELISA. (E) Pro- and mature caspase-1 and IL-1β, NLRP3, and NEK7 proteins in whole-cell lysates (WCL), and CASP1 p20 and IL-1β p17 in culture supernatants (Sup) were analyzed by immunoblotting (top). Band intensity of mature cas- pase-1 (CASP1 p20) and IL-1β (IL-1β p17) in the WCL with normalization to tubulin was shown (bottom). (F) Cleavage of GSDMD was analyzed by immunoblotting. (G) The formation of large multimeric ASC complexes was analyzed by immunoblotting.

Article Snippet: Primary antibodies recognizing NLRP3 (#15101), hemagglutinin (HA) tag (#3724), ASC/TMS1 (#67824), V5 tag (#13202), Myc tag (#2278), GSDMD (#97558), IKKβ (#2678), and caspase- 1 (#2225) were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Science advances

Article Title: Isocyanic acid-mediated NLRP3 carbamoylation reduces NLRP3-NEK7 interaction and limits inflammasome activation.

doi: 10.1126/sciadv.adq4266

Figure Lengend Snippet: Fig. 2. LACC1-produced isocyanic acid limits NLRP3 inflammasome activation. (A to G) THP-1 cells and iBMDMs without or with LACC1/Lacc1 KO were stimulated with LPS (100 ng ml−1) for 8 hours. (B to G) The LPS-primed macrophages were pretreated without or with indicated concentrations (0.5 and 1 mM) of cyanate (Cya) for 30 min and then incubated with 10 μM Nig or 5 mM ATP for another 45 min. (A) Intracellular levels of isocyanic acid (ICA) were detected by LC-MS analysis. (B to D) The release of IL-1β (B), IL-18 (C), and LDH (D) in culture supernatants was determined by ELISA. (E) Pro- and mature caspase-1 and IL-1β, NLRP3, and NEK7 proteins in WCL, and CASP1 p20 and IL-1β p17 in culture supernatants were analyzed by immunoblotting (top). Band intensity of mature caspase-1 (CASP1 p20) and IL-1β (IL-1β p17) in the WCL with normalization to tubulin was shown (bottom). (F) Cleavage of GSDMD was analyzed by immunoblotting. (G) The formation of large multimeric ASC complexes was ana- lyzed by immunoblotting. The data are presented as mean ± SD of three (A and E) or four (B to D) independent experiments. P values were calculated using one-way ANOVA (B to E). *P < 0.01, #P < 0.05.

Article Snippet: Primary antibodies recognizing NLRP3 (#15101), hemagglutinin (HA) tag (#3724), ASC/TMS1 (#67824), V5 tag (#13202), Myc tag (#2278), GSDMD (#97558), IKKβ (#2678), and caspase- 1 (#2225) were purchased from Cell Signaling Technology.

Techniques: Produced, Activation Assay, Incubation, Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Science advances

Article Title: Isocyanic acid-mediated NLRP3 carbamoylation reduces NLRP3-NEK7 interaction and limits inflammasome activation.

doi: 10.1126/sciadv.adq4266

Figure Lengend Snippet: Fig. 3. Carbamoylation sites on NLRP3 are identified, and their effects on NLRP3-NEK7 interaction are evaluated. (A) 293T cells stably expressing GSDMD were cotransfected with plasmids expressing indicated proteins. Twenty-four hours after transfection, the cells were incubated with indicated concentrations of cyanate for 30 min and then treated with Nig for another 45 min. Immunoprecipitation was performed using an anti-HA antibody followed by an immunoblotting analysis (left panel). Cyanate-mediated disruption of NLRP3-NEK7 interaction was quantified and presented as mean ± SD of three independent experiments (right panel). P values were calculated using one-way ANOVA. *P < 0.01. (B) FLAG-NLRP3 immunoprecipitated from the lysate of FLAG-NLRP3–overexpressing iBMDMs treated with 15N-labeled cyanate (1 mM) for 1 hour was separated through SDS-PAGE and stained with Coomassie brilliant blue. The indicated protein band matching the molecular weight of FLAG-NLRP3 was excised for MS analysis. HC, heavy chain; LC, light chain. (C) Schematic illustration of carbamoylation reaction (top) and carbamoylated lysine (K) residues on NLRP3 (bottom) was indicated. (D) 293T cells were cotransfected with plasmids expressing FLAG-tagged WT or indicated NLRP3 mutants, HA-NEK7, V5-ASC, and Myc- caspase-1. Twenty-four hours after transfection, the cells were incubated with cyanate (1 mM) for 30 min and then treated with Nig for another 45 min. Immunoprecipita- tion was performed using an anti-HA antibody followed by an immunoblotting analysis.

Article Snippet: Primary antibodies recognizing NLRP3 (#15101), hemagglutinin (HA) tag (#3724), ASC/TMS1 (#67824), V5 tag (#13202), Myc tag (#2278), GSDMD (#97558), IKKβ (#2678), and caspase- 1 (#2225) were purchased from Cell Signaling Technology.

Techniques: Stable Transfection, Expressing, Transfection, Incubation, Immunoprecipitation, Western Blot, Disruption, Labeling, SDS Page, Staining, Molecular Weight

Journal: Science advances

Article Title: Isocyanic acid-mediated NLRP3 carbamoylation reduces NLRP3-NEK7 interaction and limits inflammasome activation.

doi: 10.1126/sciadv.adq4266

Figure Lengend Snippet: Fig. 5. NLRP3 K593ca limits NLRP3 inflammasome activation. (A to F) NLRP3/Nlrp3-KO THP-1 cells and iBMDMs with reconstituted expression of FLAG-tagged WT h/ mrNLRP3 or h/mrNLRP3 K593R were stimulated with LPS (100 ng ml−1) for 8 hours. The LPS-primed macrophages were pretreated without or with 1 mM cyanate for 30 min and then incubated with 10 μM Nig or 5 mM ATP for another 45 min. P values were calculated using a two-tailed Student’s t test (A to C). *P < 0.01. (A to C) The release of IL-1β (A), IL-18 (B), and LDH (C) in culture supernatants was determined by ELISA. The data are presented as mean ± SD of four independent experiments. (D) Pro- and mature caspase-1 and IL-1β protein in the cell lysates were analyzed by immunoblotting. (E) Cleavage of GSDMD was analyzed by immunoblotting. (F) The formation of large multimeric ASC complexes was analyzed by immunoblotting.

Article Snippet: Primary antibodies recognizing NLRP3 (#15101), hemagglutinin (HA) tag (#3724), ASC/TMS1 (#67824), V5 tag (#13202), Myc tag (#2278), GSDMD (#97558), IKKβ (#2678), and caspase- 1 (#2225) were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Expressing, Incubation, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Science advances

Article Title: Isocyanic acid-mediated NLRP3 carbamoylation reduces NLRP3-NEK7 interaction and limits inflammasome activation.

doi: 10.1126/sciadv.adq4266

Figure Lengend Snippet: Fig. 7. Deficiency of NLRP3 K593ca exacerbates NLRP3 inflammasome activation in primary macrophages. (A to K) Isolated WT and Nlrp3 K593R mBMDMs (A) were stimulated with LPS (100 ng ml−1) for 8 hours (B to K). The LPS-primed macrophages were pretreated without or with 1 mM cyanate for 30 min and then incubated with 10 μM Nig or 5 mM ATP for another 45 min (C to I). P values were calculated using a two-tailed Student’s t test (D to F and K). N.S., not significant. *P < 0.01. (A) Flow cy- tometry analysis of mBMDMs isolated from WT or Nlrp3 K593R mouse littermates using antibodies against macrophage surface markers CD11b and F4/80. Purity of the isolated macrophages obtained from representative mouse was shown. Isolated WT mBMDMs without staining with antibodies served as a blank control. (B) NLRP3 K593ca was detected by immunoblotting. (C) Immunoprecipitation was performed using an anti-NLRP3 antibody. The immunoprecipitated protein complexes were ana- lyzed by immunoblotting. (D to F) The release of IL-1β (D), IL-18 (E), and LDH (F) in culture supernatants was determined by ELISA. The data are presented as mean ± SD of four independent experiments. (G) Pro- and mature caspase-1 and IL-1β protein in the lysates of mBMDMs were analyzed by immunoblotting. (H) Cleavage of GSDMD was analyzed by immunoblotting. (I) The formation of large multimeric ASC complexes was analyzed by immunoblotting. (J) LACC1 levels were detected by immunoblotting. (K) Intracellular isocyanic acid levels were detected by LC-MS analysis. The data are presented as mean ± SD of three independent experiments.

Article Snippet: Primary antibodies recognizing NLRP3 (#15101), hemagglutinin (HA) tag (#3724), ASC/TMS1 (#67824), V5 tag (#13202), Myc tag (#2278), GSDMD (#97558), IKKβ (#2678), and caspase- 1 (#2225) were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Isolation, Incubation, Two Tailed Test, Staining, Control, Western Blot, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Liquid Chromatography with Mass Spectroscopy

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