Journal: Journal for immunotherapy of cancer

Article Title: PARP inhibitors enhance antitumor immune responses by triggering pyroptosis via TNF-caspase 8-GSDMD/E axis in ovarian cancer.

doi: 10.1136/jitc-2024-009032

Figure Lengend Snippet: Figure 6 Caspase 8 contributes to the pyroptosis induced by PARP inhibition. Ratios of PI+ annexin V+ OVCAR8 (A), A2780 (B), and primary homologous recombination deficiency (HRD) ovarian cancer (OC) tumor cells (C) on niraparib exposure in the presence or absence of caspase inhibitors, including VX765, Z-DEVD-FMK, Z-IETD-FMK, and Z-VAD-FMK. (D) Determination of activated caspase 3 and caspase 8 levels in OVCAR8 and A2780 cells after olaparib and niraparib treatment by flow cytometry. Histological analyses of activated caspase 3 and caspase 8 in OC patient-derived xenograft (PDX) tumors (E) and triple-negative breast cancer (TNBC) PDX tumors (F) with or without niraparib exposure (n=5/6). (G) Western blotting analyses of the cleavage of caspase 8/3 and GSDMD/E in OVCAR8 and A2780 cells on niraparib treatment in the presence or absence of caspase inhibitors, including Z-DEVD-FMK, Z-IETD-FMK, and Z-VAD-FMK. Representative images of OVCAR8 (H) and A2780 cells (I) on niraparib treatment in the presence or absence of caspase inhibitors, including VX765, Z-DEVD-FMK, and Z-IETD-FMK. Mean values±SEM. *P<0.05, **p<0.01, and ***p< 0.001 by Student’s t-test in (A–C) and (E,F).

Article Snippet: VX765 (Inh vx765i) was supplied by Invivogen.

Techniques: Inhibition, Homologous Recombination, Flow Cytometry, Derivative Assay, Western Blot

Journal: Cell Death & Disease

Article Title: Rosuvastatin protects against coronary microembolization-induced cardiac injury via inhibiting NLRP3 inflammasome activation

doi: 10.1038/s41419-021-03389-1

Figure Lengend Snippet: A The H9c2 cells are stimulated with 40 ng/ml TNF-α combined with hypoxia for 12 h to active the NLRP3 inflammasome. The caspase-1 selective inhibitor VX-765 and ROS scavenger N-acetylcysteine (NAC) are added 2 h before the stimulation at 20 μM and 5 mM respectively. The pyroptotic cells are determined by Hoechst 33342/PI staining, wherein the nuclei are stained to blue by Hoechst 33342, and the pyroptotic cells are stained to red by PI. Scale bars = 50 μm. B The quantitative analysis of PI positive cells. n = 6 per group. The quantification is representative of the percentage of pyroptotic cells in 5 fields ± SEM by random. C LDH release is measured by a cytotoxicity detection LDH kit. n = 6 per group. D Cell viability is measured by a CCK-8 kit, and the optical densities (ODs) are compared among the groups. n = 6 per group. E Representative immunoblots for NLRP3, caspase-1, cleaved-caspase-1, GSDMD, GSDMD-N in H9c2 cells. F The densitometric analysis of relative protein expressions. Vinculin is used as a loading control. n = 4 per group. G Cells with active caspase-1 in each group are stained with FLICA (FAM-YVAD-FMK) probe and detected using flow cytometer. H The percentages of caspase-1 positive cells are compared among the groups. n = 6 per group. Data represent the mean ± SEM. All experiments were repeated three times. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: To demonstrate the contribution of caspase-1 and ROS to the pyroptotic cell death, the caspase-1 selective inhibitor VX-765 (Selleck Chemicals, Houston, TX, USA) and ROS scavenger NAC (MedChemExpress, Princeton, NJ, USA) were added 2 h before the stimulation of TNF-α and hypoxia at 20 μM and 5 mM respectively.

Techniques: Staining, CCK-8 Assay, Western Blot, Control, Flow Cytometry

Journal: bioRxiv

Article Title: Inhibiting ACSL1 related ferroptosis restrains MHV-A59 infection

doi: 10.1101/2021.10.14.464337

Figure Lengend Snippet: Ferroptosis inhibitor Fer-1 inhibited MHV-A59 induced syncytia and membrane damage. (A and B) Primary peritoneal macrophages (PMs) were infected with MHV-A59 at 0.05 MOI. After 2 hours of infection, cells were treated with Fer-1 (10 μM). After 24 hours of infection, cells were imaged using CytoSMART system (A) and cell confluence was evaluated using the CytoSMART website (B). (C) PMs were infected as described in (A) and were stained with propidium iodide (PI). After staining cells were imaged under fluorescence microscope. (D) PMs were infected as described in (A), followed by the treatment with Fer-1 (10 μM), z-DEVD-FMK (25 μM), VX-765 (30 μM) or GSK-872 (10 μM). Cells were stained with PI and imaged under fluorescence microscope. (E) Scale bars: For (A) and (C), 200 μm. For (D), 100 μm. Data from two independent experiments was shown. *, p < 0.05; Student’s t- test. See also Video S1.

Article Snippet: VX-765 (T6090), z-DEVD-FMK (T6005), GSK-872 (T4074), and JSH-23 (T1930) were from Targetmol.

Techniques: Membrane, Infection, Staining, Fluorescence, Microscopy

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: Nature Communications

Article Title: NLRP3 phosphorylation in its LRR domain critically regulates inflammasome assembly

doi: 10.1038/s41467-021-26142-w

Figure Lengend Snippet: a Endogenous NEK7 immunoprecipitates from LPS-primed BMDMs treated with nigericin (30 min) were analyzed for NLRP3 ( Nlrp3 +/+ +beads, Nlrp3 +/+ BMDM lysate incubated with A/G-beads without anti-NEK7). b NLRP3-deficient U937 cells reconstituted with NLRP3 mutants were treated with PMA, doxycycline, LPS, and nigericin. Endogenous NEK7 immunoprecipitates were analyzed for NLRP3 (WT + IgG, lysate of U937 expressing WT NLRP3 incubated with isotype control and A/G-beads). c , d BMDMs were treated with LPS (6 h) and nigericin (30 min). Endogenous BRCC3 immunoprecipitates were analyzed for NLRP3 ( Nlrp3 +/+ +IgG, BMDM lysate incubated with IgG isotype control and A-beads; Brcc3 −/− , BRCC3 immunoprecipitates from Brcc3 −/− BMDM lysate) ( c ). NLRP3 ubiquitination was assessed by NLRP3 immunoprecipitation followed by anti-Ub WB ( Nlrp3 +/+ +beads, Nlrp3 +/+ BMDM lysate incubated with A/G-beads without anti-NLRP3) ( d ). e NLRP3-deficient U937 cells reconstituted with NLRP3 mutants were treated with PMA and doxycycline (2 μg/ml, 16 h) followed by LPS (5 h) and nigericin (20 min). Endogenous BRCC3 immunoprecipitates were analyzed for NLRP3 (WT + IgG, lysate of U937 cells reconstituted with NLRP3 WT incubated with isotype control and A-beads). f NLRP3-deficient U937 cells reconstituted with NLRP3 mutants were treated with PMA and doxycycline (2 μg/ml, 16 h) followed by LPS (50 ng/ml, 4 h) and MG132 (10 μM, 40 min), E-64d (20 μg/ml, 40 min) and VX765 (2.5 μM, 40 min) 10 min before nigericin (30 min). NLRP3 ubiquitination was assessed by NLRP3 immunoprecipitation followed by anti-Ub WB. Caspase-1 inhibitor VX765 was added to prevent pyroptosis. g NLRP3 WT and S806D mutant were expressed with Myc-BRCC3 in 293T cells in the presence or not of HA-NEK7. Myc-BRCC3 immunoprecipitates were analyzed for NLRP3 (IgG, lysate of 293T cells expressing NLRP3 WT, Myc-BRCC3 and HA-NEK7 incubated with isotype control and A/G-beads). h Immortalized WT and Nek7 −/− BMDMs were treated with LPS (6 h) and nigericin (30 min) in the presence of VX765 (2.5 μM, 15 min before nigericin). BRCC3 immunoprecipitates were analyzed for NLRP3 by WB (WT + IgG, lysates of immortalized WT BMDMs incubated with isotype control and A-beads). i , j BMDMs were treated with LPS (6 h) and nigericin (30 min) with oridonin (2 μM, 30 min before nigericin). Endogenous BRCC3 immunoprecipitates were analyzed for NLRP3 ( i ). NLRP3 ubiquitination was assessed by NLRP3 immunoprecipitation followed by anti-Ub WB ( j ). Data are one representative of three independent experiments. Molecular weights are indicated in kDa. Lys lysates, IP immunoprecipitates, Ub ubiquitin.

Article Snippet: The following reagents were used: MG132 (Sigma-Aldrich), E-64d (Enzo Life Science), VX765 (Invivogen), G5 (Merck), doxycycline (Sigma-Aldrich), LPS (O111:B4, Sigma-Aldrich), Pam3CSK4 (Invivogen), nigericin (Invivogen), ATP (Sigma-Aldrich), H-Leu-Leu-OMe (LLOMe, Santa Cruz Biotechnology), MSU (Invivogen), silica (U.S.Silica), poly(dA:dT) (Invivogen), flagellin (FLA-ST Invivogen), oridonin (Euromedex), and D4476 (Sigma-Aldrich), SP600125 (Selleck Chemical).

Techniques: Incubation, Expressing, Control, Ubiquitin Proteomics, Immunoprecipitation, Mutagenesis

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