Journal: bioRxiv

Article Title: OLT1177 (Dapansutrile) inhibits Gasdermin D-dependent IL-1β Release and Pyroptotic Cell Death in Bone Marrow-derived Macrophages

doi: 10.64898/2026.05.31.729061

Figure Lengend Snippet: BMDMs from WT mice were treated with either media alone, lipopolysaccharide, LPS alone, LPS plus nigericin (LPS + NIG), or LPS + NIG plus OLT1177 (NLRP3 inhibitor). Nigericin (10 μM) was added to BMDMs after 3.5 hours of stimulation and cytokines were measured in the culture supernatants at 4h. IL-1β (A), CCL3 (B), and MPO (C) were measured in the culture supernatants by the R&D Duo set ELISA kit. Data are presented as mean ± SEM from three independent biological replicates (BMDMs derived from three independent mice). ****P< .0001.

Article Snippet: After 3.5 hours, the NLRP3 inflammasome formation was induced with 10 μM nigericin (InvivoGen, San Diego, CA) for 30 minutes.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: bioRxiv

Article Title: OLT1177 (Dapansutrile) inhibits Gasdermin D-dependent IL-1β Release and Pyroptotic Cell Death in Bone Marrow-derived Macrophages

doi: 10.64898/2026.05.31.729061

Figure Lengend Snippet: A) BMDMs were treated with lipopolysaccharide, LPS (1μg/mL) and LPS plus nigercin (NIG). Representative western blots show OLT1177 inhibits GSDMD cleavage to GSDMD-N in a dose-dependent manner. (B, C) Densitometric analysis of the GSDMD-N and IL-1β bands from . All concentrations of OLT1177 significantly reduce both GSDMD-N and IL-1β in a dose-dependent manner. D) Western blot shows NLRP3 and β-actin expression in BMDMs. Data are representative of three independent biological replicates (BMDMs derived from three independent mice). ****P< 0.0001.

Article Snippet: After 3.5 hours, the NLRP3 inflammasome formation was induced with 10 μM nigericin (InvivoGen, San Diego, CA) for 30 minutes.

Techniques: Western Blot, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: OLT1177 (Dapansutrile) inhibits Gasdermin D-dependent IL-1β Release and Pyroptotic Cell Death in Bone Marrow-derived Macrophages

doi: 10.64898/2026.05.31.729061

Figure Lengend Snippet: LPS induces cell activation and expression of the NLRP3 gene. Nigericin is a polyether antibiotic and acts as a potassium ionophore, causing NLRP3 inflammasome activation. Caspase-1 cleaves pro-IL-1β and GSDMD to IL-1β and GSDM-N, respectively. Mature IL-1β then exits the cells through the GSDMD-N pores.

Article Snippet: After 3.5 hours, the NLRP3 inflammasome formation was induced with 10 μM nigericin (InvivoGen, San Diego, CA) for 30 minutes.

Techniques: Activation Assay, Expressing

Journal: bioRxiv

Article Title: Opposing roles of GSDMD and NINJ1 shape early IL-1β–dependent host defense during Toxoplasma gondii infection

doi: 10.64898/2026.05.28.728484

Figure Lengend Snippet: (A and B) Bone marrow–derived macrophages (BMDMs; 2 × 10⁵ cells/well) from C57BL/6 (WT) and NINJ1 ⁻/⁻ were primed with LPS (200 ng/mL) for 3 h and subsequently stimulated with nigericin (10 µM) for 1 h as a positive control for inflammasome activation. (C-G) Alternatively, Bone marrow–derived macrophages (BMDMs; 2 × 10⁵ cells/well) from C57BL/6 (WT) and NINJ1 ⁻/⁻ mice were infected with T. gondii RH-YFP tachyzoites at a multiplicity of infection (MOI) of 2 for 18 h. After infection, cell culture supernatants were collected and cells were fixed with PFA and stained with DAPI. (A) Cell death was evaluated by measuring LDH release in culture supernatants. (B) IL-1β secretion was measured in culture supernatants by ELISA. (C) Representative immunofluorescence images of infected macrophages. (D) Parasite burden was quantified as the ratio between the YFP⁺ parasite area and the number of macrophages. (E) IL-1β secretion was measured in culture supernatants by ELISA. (F) The total number of cells remaining adherent in each well was quantified from the immunofluorescence images using ImageJ. (G) Cell death was evaluated by measuring LDH release in culture supernatants. The data shown are representative of three independent experiments performed with at least technical triplicates, except for panels A, F, and G, which are representative of two independent experiments. Statistical analysis was performed using Student’s t -test or two-way ANOVA. p < 0.05, p < 0.01, p < 0.001, p < 0.0001; ns, not significant.

Article Snippet: As a positive control for inflammasome activation, cells were primed with LPS (200 ng/mL) (InvivoGen, San Diego, CA, USA) for 3 h and subsequently treated with the agonist nigericin (10 μM) (InvivoGen) for 60 min. All infections and stimuli were performed in R3% medium, except for Western blot assays, which were conducted using Opti-MEM (Gibco) supplemented with 0.029 mM sodium bicarbonate (Sigma-Aldrich), 0.23 mM streptomycin (Sigma-Aldrich), and 0.25 mM penicillin (Sigma-Aldrich), pH 7.1.

Techniques: Derivative Assay, Positive Control, Activation Assay, Infection, Cell Culture, Staining, Enzyme-linked Immunosorbent Assay, Immunofluorescence