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

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

Journal: Cell Death and Differentiation

Article Title: RIPK3 cleavage is dispensable for necroptosis inhibition but restricts NLRP3 inflammasome activation

doi: 10.1038/s41418-024-01281-x

Figure Lengend Snippet: a BMDMs were primed for 3 h with 25 ng ml −1 LPS, then treated for 2.5 h or 5 h with 500 nM Smac-mimetic compound A. Both cell lysates and supernatants were analysed by western blot. Results are representative of 3 independent experiments performed with 3 independent biological replicates. b Cell death of BMDMs monitored by time-lapse imaging of propidium iodide (PI) staining over 6 h upon priming for 3 h with 25 ng ml −1 LPS, followed by treatment using 500 nM Smac-mimetic compound A+/− MCC950 (5 μM) or Vx765 (10 μM). Data are represented as mean + SEM of N = 3 independent biological replicates per genotype. Graphs are representative of 3 independent experiments performed with 3 independent biological replicates each time. c BMDMs were primed for 3 h with 25 ng ml −1 LPS and then treated for 2.5 h with 500 nM compound A+/− MCC950 (5 μM) or Vx765 (10 μM). Cell supernatants were assayed for IL-1β by ELISA after treatment with indicated compounds. Data are represented as mean + SEM of N = 3 independent biological replicates per genotype. Graphs are representative of 3 independent experiments performed with 3 independent biological replicates each time. d Mouse littermates were challenged with either LPS (2 mg kg −1 ) or poly(I:C) (50 μg) via intraperitoneal injection. Mouse serum was collected 2 h after treatment and assayed for IL-1β by ELISA. Data are represented as mean + SEM of N = 8 mice (LPS) or N = 9 mice (poly(I:C)) per genotype performed twice independently. c , d ** P < 0.01, *** P < 0.005.

Article Snippet: Results are representative of 3 independent experiments performed with 3 independent biological replicates. b Cell death of BMDMs monitored by time-lapse imaging of propidium iodide (PI) staining over 6 h upon priming for 3 h with 25 ng ml −1 LPS, followed by treatment using 500 nM Smac-mimetic compound A+/− MCC950 (5 μM) or Vx765 (10 μM).

Techniques: Western Blot, Imaging, Staining, Enzyme-linked Immunosorbent Assay, Injection