Journal: Frontiers in Immunology

Article Title: Mitochondrial damage and IL-1β production in monocytes caused by Neospora caninum infection are mediated by dense granule protein 7 and prohibitins

doi: 10.3389/fimmu.2025.1408992

Figure Lengend Snippet: IL-1β and TNF-α production in THP-1 cells and inflammasome-reconstructed 293T cells. (A) THP-1 cells were infected with the parental strain Nc1 or the NcGRA6-, NcGRA7-, or NcGRA14-deficient (KO) parasites at a multiplicity of infection (MOI) of 2.5 or treated with medium only (mock). At 20 h postinfection, the culture supernatants were collected for analysis. (B) 293T cells were transfected with inflammasome-reconstruction plasmids encoding NLRP3, ASC, procaspase-1, and pro-IL-1β, together with NcGRA7 cDNA or an empty vector. Untransfected 293T cells were used as a negative control (no plasmid). At 20 h posttransfection, the culture supernatants were collected for analysis. (C–H) THP-1 cells were pretreated with 10 μM MCC950 (an NLARP3 inhibitor), 100 μM VX765 (a CASP1 inhibitor), and 18 μM SN50 (an NF-κB inhibitor) for 2 hr and then infected with the Nc1 strain of N. caninum at a MOI of 2.5 or treated with medium only (mock). At 20 h postinfection, the culture supernatants were collected for analysis. Each value represents the mean ± SD of 4 replicates (technical replicates) in one representative experiment. Each experiment (biological replicate) was repeated two (G, H) , three (A, D, E, F) and four times (B, C) . Statistically significant differences according to one-way ANOVA or two-way ANOVA and a Tukey–Kramer post hoc analysis (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: MCC950 (an inhibitor of NLRP3 inflammasome activation by the inhibition of IL-1β release; Cayman Chemical), VX765 (an inhibitor of NLRP3 inflammasome activation by the inhibition of caspase 1 and IL-1β cleavage and release; LKT Laboratories), SN50 (an inhibitor of the nuclear transcription factor κB; Selleck, Houston, TX, USA), a carbonyl cyanide m-chlorophenyl hydrazone (CCCP, a uncoupling agent for oxidative phosphorylation that inhibits mitochondrial function; FUJFILM Wako Pure Chemical Corporation), rocaglamid A (Cayman, Ann Arbor, MI, USA) with biochemical research grades were used in this study.

Techniques: Infection, Transfection, Plasmid Preparation, Negative Control

Journal: Cell Death Discovery

Article Title: Osteocytic Lipocalin-2 regulates bone formation locally through iron-dependent ferroptosis and Wnt suppression

doi: 10.1038/s41420-026-02956-9

Figure Lengend Snippet: qPCR analysis of Lcn2 and osteoblast and osteocyte markers ( Phex, Runx2, Dmp1, Fgf23, Sost ) during osteogenic differentiation of A BMSCs and B OCY454 osteocyte-like cells. p < 0.05 vs. week 0, n = 4 biological replicates per time point. C – I OCY454 cells were treated with recombinant murine LCN2 (rmLCN2, 100 ng/mL) ± the ferroptosis inhibitor Deferoxamine (DFO, 100 µM) for 24 h. C Quantification of FerroOrange staining showing elevated Fe²⁺ accumulation; D , E total and Fe³⁺ iron concentrations; F , G DCFDA fluorescence showing increased ROS; H , I C11-BODIPY fluorescence indicating lipid peroxidation. DFO co-treatment rescued all parameters. n = 3–4 biological replicates per group. J Annexin V/PI flow-cytometric analysis showing increased cell death with LCN2, fully rescued by DFO but not by inhibitors of apoptosis (DEVD), necroptosis (Necrostatin-1), or pyroptosis (VX-765), confirming ferroptosis as the predominant mode of cell death. K – P Loss-of-function validation in OCY454 cells transfected with Lcn2 shRNA or scrambled control. K , L , M qPCR and Western blot confirming knockdown efficiency; N , O , P flow-cytometric analysis showing reduced ROS (DCFDA, N), lipid peroxidation (C11-BODIPY, O), and cell death (Annexin V/PI, P) following Lcn2 silencing. n = 3–4 biological replicates per group. Data are presented as mean ± SD. * p < 0.05 vs. control or scrambled shRNA; $ p < 0.05 vs. LCN2 alone; one-way ANOVA with Newman-Keuls post hoc test or Student’s t test as appropriate. Data in ( A – P ) are representative of three independent experiments.

Article Snippet: 16–18 h post-transfection, cells were treated with rmLCN2 (L) or control (C) for an additional 24 h. For iron overload, OCY454 cells were cultured in the presence of ferric ammonium chloride (FAC, 250 μM, Sigma; Cat# 158040) for 24 h, while rmLCN2 treatment was reduced to 6 h. To assess cell death pathways, cells were treated with rmLCN2 (100 ng/mL) in the presence of selective inhibitors: the ferroptosis inhibitor deferoxamine (DFO, 100 μM, Tocris Cat# 14595), the necroptosis inhibitor necrostatin-1 (Nec-1, 20 μM, Tocris Cat# 11658), the apoptosis inhibitor DEVD (10 μM, Tocris Cat# 14414), and the pyroptosis inhibitor VX-765 (10 μM, Tocris Cat#28825).

Techniques: Recombinant, Staining, Fluorescence, Biomarker Discovery, Transfection, shRNA, Control, Western Blot, Knockdown

Journal: EMBO Molecular Medicine

Article Title: Nanobodies dismantle post‐pyroptotic ASC specks and counteract inflammation in vivo

doi: 10.15252/emmm.202115415

Figure Lengend Snippet: A–D (A, B) Human IL‐1β (hIL‐1β) concentrations in cell‐free supernatants of LPS‐primed (10 ng ml −1 , 150 min) primary human macrophages that were left untreated, or pre‐incubated with VHH ASC or mutVHH ASC (100 µg ml −1 ), CRID3 (50 µM) or VX‐765 (50 µM) for 30 min before stimulation with (A) nigericin (10 µM), or (B) PFO (30 ng ml −1 ) for 2 h. (C‐D) Mouse IL‐1β (mIL‐1β) concentrations in cell‐free supernatants of LPS‐primed mouse BMDMs (200 ng ml −1 , 150 min), incubated with VHHs, CRID3 or VX765, before activation with nigericin (10 µM), or PFO (250 ng ml −1 ). Data is combined from two independent experiments, each performed with two donors (A, B) or mice (C, D), represented with individual symbols (4 donors or mice in total). Data is displayed as floating bars with the max/min values and mean (thicker band). E, F (E) Epifluorescence microscopy imaging and (F) quantification of ASC speck formation in BMDMs from ASC‐mCitrine (Green) transgenic mice. Cells were primed with LPS (200 ng ml −1 , 150 min), pre‐treated with VX‐765 (50 µM, 30 min), then treated with VHH ASC , VHH mASC (100 µg ml −1 ) or CRID3 (50 µM) for another 30 min before stimulation with nigericin (top), or PFO (bottom) for 2 h and finally fixed with 4% PFA. Nuclei was stained with DRAQ5 (Blue). Scale bars: 100 μm. Images in (E) are from one representative out of three independent experiments that were quantified in F. Data in F is displayed as floating bars with the max/min values and mean (thicker band). Data information: ** P < 0.005; *** P < 0.0002; **** P < 0.0001, One‐way ANOVA, multiple comparison (Tukey test).

Article Snippet: Inhibitors used were CRID3 (Tocris) and VX‐765 (Selleckchem).

Techniques: Incubation, Activation Assay, Epifluorescence Microscopy, Imaging, Transgenic Assay, Staining, Comparison

Journal: EMBO Molecular Medicine

Article Title: Nanobodies dismantle post‐pyroptotic ASC specks and counteract inflammation in vivo

doi: 10.15252/emmm.202115415

Figure Lengend Snippet: A, B Cell viability (CTB) assay on LPS‐primed primary human macrophages that were left untreated, or pre‐incubated with VHH ASC or mutVHH ASC (100 µg ml −1 ), CRID3 (50 µM) or VX‐765 (50 µM) for 30 min before being activated with (A) nigericin (10 µM), or (B) PFO (30 ng ml −1 ) for 2 h. Data is from the experiments displayed in Fig A and B. C Human IL‐1β (hIL‐1β) concentrations in cell‐free supernatants (left), and cell viability assay (right) of LPS‐primed primary human macrophages that were incubated with VHH ASC or mutVHH ASC (100 µg ml −1 ), CRID3 (50 µM) or VX‐765 (50 µM) for 30 min before being stimulated with 2.5 mM ATP. D hIL‐1β concentrations in cell‐free supernatants (top), and cell viability assay (bottom) of PMA‐differentiated THP‐1 cells treated with VHH ASC or mutVHH ASC (100 µg ml −1 ), CRID3 (10 µM) or VX‐765 (50 µM) for 30 min before 4.5 h stimulation with 250 µg ml −1 MSU crystals. E hIL‐1β concentrations in cell‐free supernatants (top), and cell viability assay (bottom) of LPS‐primed primary human macrophages that were incubated with VHH ASC or mutVHH ASC (100 µg ml −1 ), CRID3 (50 µM) or VX‐765 (50 µM) for 30 min before being stimulated with 0.1 µg ml −1 /0.5 µg ml −1 mixture of LFn‐BsaK and PA for 2 h. F hIL‐1β concentrations in cell‐free supernatants (top), and cell viability assay (bottom) of Pam3CysK4‐primed (1 µg ml −1 ) primary human CD14 + monocytes that were incubated with VHH ASC or mutVHH ASC (100 µg ml −1 ), or VX‐765 (50 µM) for 30 min before being stimulated with 1 µg ml −1 TcdA. G hIL‐1β concentrations in cell‐free supernatants (top), and cell viability assay (bottom) of keratinocyte cells (N‐TERT) that were treated with VHH ASC or mutVHH ASC (100 µg ml −1 ), or VX‐765 (50 µM), then directly stimulated with 30 µM Val‐boroPro (VbP) for 22 h. H hIL‐1β concentrations in cell‐free supernatants (top), and cell viability assay (bottom) of PMA‐differentiated THP‐1 cells treated with IFNγ (500 U ml −1 ) for 16 h and that were incubated with VHH ASC or mutVHH ASC (100 µg ml −1 ), CRID3 (10 µM) or VX‐765 (50 µM) for 30 min before 2 h stimulation with 1 µg ml −1 poly(dA:dT) in complex with Lipofectamine 2000. Data information: Data is representative of either two independent experiments, each run with one to two donors (A–C, E, F, 3 or 4 donors in total) or at least 3 independent experiments (D, G, H). Each symbol represents one donor or independent experiment. ns P > 0.05; * P < 0.05; ** P < 0.005; *** P < 0.0002; **** P < 0.0001, One‐way ANOVA, multiple comparison (Tukey test). Data is displayed as floating bars with the max/min values and mean (thicker band).

Article Snippet: Inhibitors used were CRID3 (Tocris) and VX‐765 (Selleckchem).

Techniques: CtB Assay, Incubation, Viability Assay, Comparison

Journal: EMBO Molecular Medicine

Article Title: Nanobodies dismantle post‐pyroptotic ASC specks and counteract inflammation in vivo

doi: 10.15252/emmm.202115415

Figure Lengend Snippet: A Specificity of different single‐domain antibodies (VHHs) probed by ELISA. Recombinant murine ASC as an eGFP fusion or eGFP alone (GFP‐LPETG) as control, were coated onto ELISA plates at 1 µg ml −1 /well. Wells were incubated with HA‐tagged VHHs (100 nM), anti‐HA‐tag mouse mAb coupled to HRP, and the HRP substrate TMB. Binding was quantified by measuring the absorbance at 450 nm. B, C Lysates of HEK 293T cells transiently expressing HA‐tagged VHH mASC or VHH ASC and the indicated bait proteins fused to Renilla luciferase were used to immunoprecipitate VHHs with immobilized anti‐HA antibody. Renilla luciferase activity of the co‐immunoprecipitated proteins was measured and normalized to the input luciferase. Data represents mean values ± SEM from three independent experiments. D, E Cell viability (CTB) assay on LPS‐primed (200 ng ml −1 ) primary mouse BMDMs that were left untreated, or pre‐incubated with VHH ASC or VHH mASC (100 µg ml −1 ), CRID3 (50 µM) or VX‐765 (50 µM) for 30 min before being activated with (D) nigericin (10 µM), or (E) PFO (250 ng ml −1 ) for 2 h. Data is from the experiments displayed in Fig C and D. Data is displayed as floating bars with the max/min values and mean (thicker band). F Mouse IL‐1β (mIL‐1β) concentrations in cell‐free supernatants (left), and cell viability assay (right) of LPS‐primed mouse BMDMs incubated with VHHs, CRID3 or VX–765, before stimulation with 0.1 µg ml −1 /0.5 µg ml −1 mixture of LFn‐BsaK and PA for 2 h. Each symbol represents one mouse. Data is displayed as floating bars with the max/min values and mean (thicker band). Data information: ns P > 0.05; *** P < 0.001; **** P < 0.0001, Two‐way (A, C) or One‐way (B, D–F) ANOVA, multiple comparison (Tukey test).

Article Snippet: Inhibitors used were CRID3 (Tocris) and VX‐765 (Selleckchem).

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Control, Incubation, Binding Assay, Expressing, Luciferase, Activity Assay, Immunoprecipitation, CtB Assay, Viability Assay, Comparison

Journal: EMBO Molecular Medicine

Article Title: Nanobodies dismantle post‐pyroptotic ASC specks and counteract inflammation in vivo

doi: 10.15252/emmm.202115415

Figure Lengend Snippet: THP‐1 cells expressing a Dox‐inducible CRISPR‐Cas9 cassette targeting GSDMD were left untreated (–), or treated with 1 µg ml −1 Dox for one or two cycles of 72 h (1×, or 2× respectively). Immunoblot analysis of GSDMD expression following the indicated course of Dox treatment and PMA‐differentiation, as indicated. Data is from one representative of two independent experiments. IL‐1β concentration or percentage of LDH released into cell‐free supernatants of PMA‐differentiated THP‐1 cells that were treated with VHH ASC (200 µg ml −1 ) or CRID3 (25 µM) for 30 min prior to stimulation with nigericin (10 µM, left panels) or PFO (30 ng ml −1 , right panels) for 2 h. Data is average of experimental duplicates from three independent experiments, each represented by a different symbol. ns P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, Two‐way ANOVA, multiple comparison (Tukey test). Data is displayed as floating bars with the max/min values and mean (thicker band). Live confocal imaging of PMA‐differentiated and nigericin‐treated (10 µM) THP‐1 cells expressing human ASC‐GFP (green) in the presence of AlexaFluor647‐labeled VHH ASC (VHH ASC ‐AF647, 10 µg ml −1 , cyan) in the medium. Cells were either left untreated (–) or incubated with VX‐765 (50 µM) for 1 h prior to nigericin stimulation. Nuclei were stained with Hoechst 34580 (magenta). Scale bar: 50 µm. Data is from one representative out of two independent experiments. Source data are available online for this figure.

Article Snippet: Inhibitors used were CRID3 (Tocris) and VX‐765 (Selleckchem).

Techniques: Expressing, CRISPR, Western Blot, Concentration Assay, Comparison, Imaging, Labeling, Incubation, Staining

Journal: Cell reports

Article Title: Protein folding stress potentiates NLRP1 and CARD8 inflammasome activation

doi: 10.1016/j.celrep.2022.111965

Figure Lengend Snippet: (A) Schematic of the experiment to assess DPP9-CARD8 ternary complex displacement in cells. (B) HEK 293TCASP1+GSDMD cells were transiently transfected plasmids encoding dTAG-CARD8ZUC and the isolated FIINDSA 48 h prior to treatment with dTAG-13 (500 nM), VbP (10 μM), MeBs (10 μM), or the combination for 6 h. Samples were collected and analyzed by immunoblotting and LDH release. (C) DPP8−/−/DPP9−/− THP-1 cells were treated with MeBs (10 μM) and/or bortezomib (Bort., 10 μM) for 8 h before LDH and immunoblot analyses. (D and E) CARD8−/− THP-1 cells stably containing doxycycline (DOX)-inducible CARD8 WT, E274R, and/or S297A were induced with 100 ng/mL DOX for 16 h prior to treatment with VbP (10 μM), MeBs (10 μM), VX765 (50 μM), Bort. (10 μM), and/or MG132 (10 μM) for 6 h. Samples were then collected for LDH release and immunoblot analyses. Note in (D) that immunoblot and LDH analyses were performed separately. Data are means ± SEM of three biological replicates. All data, including immunoblots, are representative of three or more independent experiments. ***p < 0.001, **p < 0.01, *p < 0.05 by two-sided Student’s t test. n.s., not significant. See also Figure S2.

Article Snippet: VX765 , Cayman , 28825.

Techniques: Transfection, Isolation, Western Blot, Stable Transfection

Journal: Cell reports

Article Title: Protein folding stress potentiates NLRP1 and CARD8 inflammasome activation

doi: 10.1016/j.celrep.2022.111965

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: VX765 , Cayman , 28825.

Techniques: Recombinant, LDH Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Viability Assay, Cytotoxicity Assay, Software

Journal: Cell reports

Article Title: Protein folding stress potentiates NLRP1 and CARD8 inflammasome activation

doi: 10.1016/j.celrep.2022.111965

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: VX765 , Cayman , 28825.

Techniques: Recombinant, LDH Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Viability Assay, Cytotoxicity Assay, Software