caspase 4 inhibitor vx 765  (InvivoGen)

Journal: The FASEB Journal

Article Title: Timing of Glucocorticoid Treatment Dictates Glucocorticoid Receptor Actions Modulating the NLRP3‐Inflammasome Activation in Macrophages

doi: 10.1096/fj.202504083R

Figure Lengend Snippet: Glucocorticoids modulate inflammation and pyroptosis by regulating late NLRP3‐inflammasome activation through ACOD1 and iNOS. (A) Schematic representation of the experimental setup for conventional NLRP3‐inflammasome activation (upper), and late NLRP3‐inflammasome activation regulated by Dex and MCC950 (lower). (B) and (C) Quantification of IL‐1β concentration determined by ELISA in the supernatant of WT and GRKO BDMDs stimulated with LPS for 3 h (conventional) and 24 h (late), with Dex and vehicle added during the final 2 and 8 h of treatment, followed by ATP (5 mM) or Nigericin (10 μM) ( n = 3). (D) Immunoblot analysis depicting Dex effects on the protein levels of ACOD1, iNOS, NLRP3, pro‐IL‐1β, Caspase‐1 and GSDM‐D during late LPS‐induced NLRP3‐inflammasome activation and using ATP as signal 2 in WT and GRKO BMDMs lysates, detected with specific antibodies using western blotting. (E) Densitometry values of the immunoreactive bands quantified for protein content, normalized to β‐actin as depicted in (D) ( n = 4). (F) Nitrite levels in the supernatants of WT and GRKO BMDMs as depicted in (D) determined by colorimetric assay ( n = 4). (G) Assessment of lactate dehydrogenase (LDH) activity in the supernatants of WT and GRKO BMDMs as depicted in (D) ( n = 4). (H) Immunoblot analysis depicting the protein levels of NRF2 as depicted in (D) in presence of the proteasome inhibitor MG‐132 (2 μM) 6 h before harvesting cells ( n = 3). (I) Immunoblot analysis depicting the protein levels of HIF‐1α as depicted in (D) in presence of the prolyl‐hydroxylase inhibitor Roxadustat (RXD; 10 μM) 2 h before adding Dex ( n = 3). (J) Quantification of IL‐1β concentration determined by ELISA in the supernatant of WT BDMDs as depicted in (H) and (I) ( n = 3). (K) Assessment of lactate dehydrogenase (LDH) activity in supernatants of WT BMDMs subjected to the conditions described in (H) and (I) ( n = 3). Data represented as mean ± SEM. Blots shown are representative of a minimum of 3 independent experiments. Statistical analysis was performed using 2‐way ANOVA with Sidak's multiple‐comparison test (B–I); one‐way ANOVA with Tukey's multiple‐comparison test (J) and (K). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Lipopolysaccharide from E. coli 0111: B4 strain (LPS‐EB), NLRP3 inhibitor MCC950, Caspase‐1 inhibitor Ac‐Y‐VAD‐cmk, 4‐octyl‐itaconate (4‐OI), adenosine 5′‐triphosphate disodium salt (ATP), nigericin, and monosodium urate (MSU) crystals were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Activation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Colorimetric Assay, Activity Assay, Comparison

Journal: Immunity, Inflammation and Disease

Article Title: Kaempferitrin Attenuates Lipopolysaccharide‐Induced Cardiac Dysfunction Through Suppression of the NF‐κB/NLRP3 Signaling Pathway

doi: 10.1002/iid3.70323

Figure Lengend Snippet: Kae inhibited LPS‐induced cardiomyocyte inflammation and the activation of NF‐κB/NLRP3/pyroptosis pathway in vitro. (A–C) Transcriptome sequencing of myocardial tissue from LPS group and LPS + Kae group. It showed a volcano plot (A), GSEA analysis of NF‐κB pathway (B), and a heat map of genes involved in pyroptosis and NF‐κB signaling pathway (C), N = 3. (D) CCK8 assay evaluating the appropriate concentration of Kaempferitrin Kae). (E) The brief experimental setup of the in vitro experiment. After maltreatment with Kaempferitrin for 2 h, cardiomyocytes were then exposed to LPS (1 μg/L) for 12 h. (F) The relative RNA levels of Il‐1β, Il‐6 , and Tnf‐ α in H9c2 cells. (G) The relative protein levels of NLRP3, p65, p‐p65, Ik‐Bα, p‐Ik‐Bα and GAPDH in H9c2 cell were detected using western blot assay. (H) The densitometric quantification of NLRP3, p‐Ik‐Bα/Ik‐Bα and p‐P65/P65 in H9c2 cells. (I) The relative mRNA levels of Il‐1β , Il‐6 , and Tnf‐α in AC16 cells. (J) The relative protein levels of NLRP3, p65, p‐p65, Ik‐Bα, p‐Ik‐Bα, and GAPDH in AC16 cell were detected using western blot assay. (K) Densitometric quantification of NLRP3, p‐Ik‐Bα/Ik‐Bα, and p‐P65/P65 in AC16 cells. (L, M) The relative protein levels of Cleaved‐GSDMD, Cleaved‐caspase1, and GAPDH in H9c2 cells (L) and AC16 cells (M) were detected using western blot assay. N = 3. (One‐way analysis of variance [ANOVA] followed by Bonferroni post hoc test for comparing multiple groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant).

Article Snippet: To evaluate the impact of pyroptosis in Kae‐mediated cardioprotection against LPS‐induced cardiotoxicity, the caspase‐1 inhibitor VX765 (MCE, #HY‐13205, 25 μM) [ ] was utilized in accordance with the manufacturer's instructions.

Techniques: Activation Assay, In Vitro, Sequencing, CCK-8 Assay, Concentration Assay, Western Blot

Journal: Immunity, Inflammation and Disease

Article Title: Kaempferitrin Attenuates Lipopolysaccharide‐Induced Cardiac Dysfunction Through Suppression of the NF‐κB/NLRP3 Signaling Pathway

doi: 10.1002/iid3.70323

Figure Lengend Snippet: Kaempferitrin may ameliorate LPS‐induced cardiac injury by inhibiting pyroptosis via the NF‐κB/NLRP3 pathway in vivo . (A) The relative protein levels of NLRP3, P65, p‐P65, IkBα, p‐IkBα, and GAPDH in the heart tissue were detected using western blot assay. (B)The relative protein levels of GSDMD, Cleaved‐caspase1, and GAPDH in the heart tissue were detected using western blot assay. C‐E, Densitometric quantification of NLRP3 (C), p‐IkBα/IkBα (D), and PP65/P65 (E). (F, G) Densitometric quantification of Cleaved‐GSDMD and Cleaved‐caspase 1. (H, I) Levels of IL‐1β (H), IL‐18 (I) in heart tissue detected by ELISA assay. N = 6. (One‐way analysis of variance [ANOVA] followed by Bonferroni post hoc test for comparing multiple groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant).

Article Snippet: To evaluate the impact of pyroptosis in Kae‐mediated cardioprotection against LPS‐induced cardiotoxicity, the caspase‐1 inhibitor VX765 (MCE, #HY‐13205, 25 μM) [ ] was utilized in accordance with the manufacturer's instructions.

Techniques: In Vivo, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Immunity, Inflammation and Disease

Article Title: Kaempferitrin Attenuates Lipopolysaccharide‐Induced Cardiac Dysfunction Through Suppression of the NF‐κB/NLRP3 Signaling Pathway

doi: 10.1002/iid3.70323

Figure Lengend Snippet: Kaempferitrin improves LPS‐induced myocardial inflammatory damage by inhibiting the NF‐κB/NLRP3/pyroptosis pathway. After pretreatment with Kaempferitrin, VX765 or Kaempferitrin+VX765 for 2 h, cardiomyocytes were then exposed to LPS (1 μg/mL) for 12 h. (A) The relative protein levels of GSDMD, Cleaved‐caspase1 and GAPDH in H9c2 cell were detected using western blot assay. The densitometric quantification of Cleaved‐GSDMD (B), and Cleaved‐Caspase1 (C) in H9c2 cells. (D) The relative mRNA levels of Il‐1β , Il‐6 , and Tnf‐α in H9c2 cells. (E) The relative protein levels of GSDMD, Cleaved‐caspase1, and GAPDH in AC16 cell were detected using western blot assay. The densitometric quantification of Cleaved‐GSDMD (F) and Cleaved‐Caspase1 (G) in AC16 cells (H). N = 3. (One‐way analysis of variance [ANOVA] followed by Bonferroni post hoc test for comparing multiple groups. * p < 0.05, ** p < 0.01, ns = not significant).

Article Snippet: To evaluate the impact of pyroptosis in Kae‐mediated cardioprotection against LPS‐induced cardiotoxicity, the caspase‐1 inhibitor VX765 (MCE, #HY‐13205, 25 μM) [ ] was utilized in accordance with the manufacturer's instructions.

Techniques: Western Blot

Journal: Immunity, Inflammation and Disease

Article Title: Kaempferitrin Attenuates Lipopolysaccharide‐Induced Cardiac Dysfunction Through Suppression of the NF‐κB/NLRP3 Signaling Pathway

doi: 10.1002/iid3.70323

Figure Lengend Snippet: NLRP3 is required for Kae to defend against cardiotoxicity induced by LPS in vivo. The WT mice and NLRP3 knockout mice were exposed to LPS (10 mg/kg) for 12 h. (A) Representative transthoracic echocardiography M‐model images from each group. (B) EF% and (C) FS% of the mice in each group. (D, E) Serum levels of CK‐MB and LDH in mice from each group. (F) Representative images of HE staining and TUNEL staining for heart tissue. Scale bar = 50 μm. N = 6. (G) The relative protein levels of Cleaved‐GSDMD, Cleaved‐caspase1, and GAPDH in the heart tissue were detected using western blot assay. Densitometric quantification of C‐GSDMD (H) and C‐Caspase 1 (I) were shown. N = 6. (One‐way analysis of variance [ANOVA] followed by Bonferroni post hoc test for comparing multiple groups. * p < 0.05, ** p < 0.01, **** p < 0.0001, ns = not significant).

Article Snippet: To evaluate the impact of pyroptosis in Kae‐mediated cardioprotection against LPS‐induced cardiotoxicity, the caspase‐1 inhibitor VX765 (MCE, #HY‐13205, 25 μM) [ ] was utilized in accordance with the manufacturer's instructions.

Techniques: In Vivo, Knock-Out, Staining, TUNEL Assay, Western Blot

Journal: bioRxiv

Article Title: Local albumin excess exacerbates Candida albicans-induced inflammasome activation linked with hyperinflammation during vulvovaginal candidiasis

doi: 10.64898/2026.01.22.700771

Figure Lengend Snippet: (A) Overview of NLRP3 inflammasome priming and activation. DAMPs = damage-associated molecular patterns. TLR = Toll-like receptor. PAMPs = pathogen-associated molecular pattern. (B) IL-1β and IL-18 release by hMDMs ( n = 14 donors). (C) IL-1β release by mBMDMs ( n = 10). (D) IL-1β release by hMDMs in the presence of the inflammasome inhibitors KCl ( n = 8 donors) or VX-765 ( n = 9 donors). (E) Percentage ASC-positive mBMDMs ( n = 3 uninfected , n = 4 infected). (F, G) IL-1β release of mBMDMs deficient in the NLRP3 receptor ( Nlrp3 −/− ), the adapter proteins ASC ( Pycard −/− ) or the proteolytic enzyme caspase 1 ( Casp1 −/− / Casp11 −/− ) ( n = 4) or deficient in gasdermin D ( Gsdmd −/− ) (G). Bars represent the mean + SEM with dots as individual biological replicates: Individual donors (B, D) or mice (C, E - G) from at least three independently conducted experiments. Statistical significance was determined using a two-way ANOVA including with Holm-Šídák post-hoc test. * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001

Article Snippet: For inhibitor experiments, Anakinra (recombinant human IL-1Ra, 10 μg/mL, Kineret), potassium chloride (25 mM; Merck) or the caspase-1 inhibitor VX-765 (50 μg/mL; Invivogen) were added 1 h prior to infection.

Techniques: Activation Assay, Infection