z yvad fmk  (Santa Cruz Biotechnology)

Journal: Advanced Science

Article Title: Membrane‐Active Peptide Protects Against Inflammation by Targeting NLRP3 Activation at the Trans‐Golgi Network

doi: 10.1002/advs.76587

Figure Lengend Snippet: Pre‐incubation with Pep19‐2.5 inhibits nigericin‐ and MSU crystal‐induced IL‐1β secretion. (A) Primary monocytes were primed with 1 µg/mL Pam 3 CSK 4 for 3 h and then stimulated with 10 µM nigericin or 200 µg/mL MSU crystals for 3 h. Pep19‐2.5 was added 30 min before priming at increasing concentrations ranging from 0.4 to 18 µM. Supernatants were analyzed for IL‐1β by ELISA. Stimulus‐induced IL‐1β secretion was set to 100%. Mean ± SEM ( n = 4 biologically independent experiments for nigericin, n = 2 biologically independent experiments for MSU crystals). One‐sample t ‐test against 100%. (B) hMDMs were primed with 1 µg/mL Pam 3 CSK 4 for 4 h and then stimulated with 5 µM nigericin for 2 h. Pep19‐2.5 was added at the indicated concentrations 30 min before priming. Supernatants were analyzed for IL‐1β by ELISA. Stimuli‐induced IL‐1β secretion was set to 100%. Mean + SEM ( n = 4 biologically independent experiments). One‐sample t ‐test against 100%. (C) THP‐1 macrophages were primed and stimulated as described in (A). For inhibition experiments, 18 µM Pep19‐2.5 was added 30 min before priming. Supernatants were analyzed for IL‐1β by ELISA. Stimulus‐induced IL‐1β secretion was set to 100%. Mean + SEM ( n = 4 biologically independent experiments for nigericin, n = 3 biologically independent experiments for MSU crystals). One‐sample t ‐test against 100%. (D and E) THP‐1 macrophages were primed and treated with NLRP1 (D) or AIM2 (E) activators. Supernatants were analyzed for IL‐1β by ELISA. Stimulus‐induced IL‐1β secretion was set to 100%. Mean + SEM ( n = 3 biologically independent experiments). One‐sample t ‐test against 100%. (F–H) Protein expression of pro‐IL‐1β (F), full‐length (G), and cleaved GSDMD (H) were analyzed by Western blot. Basal, Pam 3 CSK 4 ‐ or nigericin‐induced expression was set to 100%. Mean + SEM ( n = 3 biologically independent experiments). One‐sample t ‐test against 100%. (I) THP‐1 macrophages were primed and stimulated after peptide incubation as described in (A). After 1 h of stimulation with nigericin, the medium was removed, and Z‐WEHD buffer was added according to the manufacturer´s protocol. Ac‐YVAD‐CHO control was subtracted, and stimulus‐induced caspase‐1 activity was normalized to 100%. Mean ± SEM ( n = 3 biologically independent experiments). One‐sample t ‐test against 100%. (J) hMDMs from healthy donors were seeded in µ‐Slides VI and incubated at 37°C in a humidified atmosphere of 5% CO 2 for 1 h. Macrophages were primed with 1 µg/mL Pam 3 CSK 4 for 3 h and stimulated with 10 µM nigericin for 1 h in the presence of fluorophore‐conjugated Rh‐Pep19‐2.5. Active caspase‐1 was stained with the caspase‐1 pseudosubstrate FLICA660‐YVAD‐FMK. Cells were washed and fixed, and nuclei were stained with Hoechst 34580. Confocal microscopy images are representative of n = 2 biologically independent experiments. Scale bar is 10 µm (upper panel) and 2 µm (zoomed in lower panel). (K) Solutions containing 0.5 µM Atto488‐conjugated Pep19‐2.5 were prepared in the presence of increasing concentrations of recombinant human caspase‐1 and incubated overnight at 4°C under gentle agitation. Microfluidic diffusional sizing (MDS) measurements were performed at room temperature to determine the hydrodynamic radius ( R h ) of fluorescently labeled particles. Dots represent the mean ± SD of replicates ( n ≥ 3) of free peptide and peptide–protein complexes, and the black line represents the best nonlinear fit according to Equation . (L) Human recombinant caspase‐1 was incubated with the caspase‐1‐specific substrate Ac‐YVAD‐AMC at the indicated concentrations of Pep19‐2.5 or 40 µM of the caspase‐1 inhibitor VX765. Enzyme activity in relative fluorescence units (RFU) was measured every 2 min at 37°C. Representative traces are means ± SEM ( n = 2 biologically independent experiments).

Article Snippet: For this purpose, Pep19‐2.5 or the specific caspase‐1 inhibitor VX765 (HY‐13205, MedChemExpress, NJ, USA) was added in triplicates to the assay buffer (pH = 7.4) consisting of 50 mM HEPES, 100 mM NaCl, 0.1% CHAPS (w/v), 1 mM EDTA, 10% glycerol (v/v), and 10 mM DTT.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Inhibition, Expressing, Western Blot, Control, Activity Assay, Staining, Confocal Microscopy, Recombinant, Gentle, Labeling, Fluorescence

Journal: Advanced Science

Article Title: Membrane‐Active Peptide Protects Against Inflammation by Targeting NLRP3 Activation at the Trans‐Golgi Network

doi: 10.1002/advs.76587

Figure Lengend Snippet: Pep19‐2.5 inhibits NLRP3‐induced ASC assembly but does not bind to NLRP3. (A) Representative Western blot of cross‐linked cytosolic pellet from primed THP‐1 macrophages that were pre‐incubated with increasing concentrations of Pep19‐2.5 and stimulated with 10 µM nigericin for 1 h ( n = 3 biologically independent experiments). (B) HEK293 ASC‐BFP cells transduced with NLRP3 were pre‐incubated with increasing concentrations of Pep19‐2.5 or MCC950 and stimulated with nigericin. ASC speck formation was determined by flow cytometry. Dot plots show mean ± SEM ( n = 3 biologically independent experiments). One‐way ANOVA followed by Dunnet's post‐test. (C) THP‐1 C1C‐EGFP macrophages were incubated with the indicated concentrations of Pep19‐2.5 for 30 min prior to priming. After priming with Pam 3 CSK 4 , 40 µM VX765 was added to limit cell death, and 10 µM nigericin was added to stimulate NLRP3 assembly for 1 h. Staining was performed using an anti‐ASC antibody and Hoechst 34580. Confocal microscopy images are representative of n = 2 biologically independent experiments, with three individual images taken for each condition. Scale bar = 20 µm. Full images are shown Figure . (D) Nuclei, ASC, and C1C specks in the images shown in (C) were quantified separately in the corresponding fluorescence channels. For each condition, three independent microscopy images were analyzed, with each data point representing a single image. Speck formation was quantified manually, and results are presented as mean + SEM. One‐way ANOVA followed by Šídák's multiple comparison post hoc analysis. (E) Melting temperature of NLRP3 NACHT alone, with the inhibitor MCC950 or Pep19‐2.5, as determined by nanoDSF ( n = 3 independent experiments). No shift upon addition of Pep19‐2.5 is observed, indicating no direct interaction of Pep19‐2.5 with the NACHT domain of NLRP3. (F) The thermal stability of the decameric NLRP3 (fl., wt) protein with and without Pep19‐2.5 was determined by measuring the hydrodynamic radius by DLS ( n = 3 independent experiments).

Article Snippet: For this purpose, Pep19‐2.5 or the specific caspase‐1 inhibitor VX765 (HY‐13205, MedChemExpress, NJ, USA) was added in triplicates to the assay buffer (pH = 7.4) consisting of 50 mM HEPES, 100 mM NaCl, 0.1% CHAPS (w/v), 1 mM EDTA, 10% glycerol (v/v), and 10 mM DTT.

Techniques: Western Blot, Incubation, Transduction, Flow Cytometry, Staining, Confocal Microscopy, Fluorescence, Microscopy, Comparison, Nano Differential Scanning Fluorimetry

Journal: Advanced Science

Article Title: Membrane‐Active Peptide Protects Against Inflammation by Targeting NLRP3 Activation at the Trans‐Golgi Network

doi: 10.1002/advs.76587

Figure Lengend Snippet: Pep19‐2.5 reduces pro‐inflammatory IL‐1β response to house dust mite extract in vitro and in vivo. (A–E) Human macrophages from heathy donors were seeded in serum‐free Opti‐MEM medium and stimulated without priming with the indicated doses of house dust mite (HDM) extract for 24 h at 37°C (A) alone, in the presence of (B) the NLRP3 inhibitor MCC950 (10 µM), (C) the TLR4 antagonist eritoran (1 µg/mL), or (D–F) Pep19‐2.5. IL‐1β release was determined from cell‐free supernatants by ELISA. Data shown in (A) mean + SD of duplicate analyses and are representative for n = 7 independent donors. Data in (B–E) were normalized to 100% for HDM in the absence of inhibitors and are shown as mean + SEM of (B, C, E, F): n = 5 and (D): n = 3 independent experiments with cells from different healthy donors. Statistical significance against HDM alone was analyzed by a two‐sided t ‐test. (G) Treatment protocol for HDM‐induced allergic asthma mouse model. (H) Airway resistance in response to methacholine inhalation, (I) inflammatory cell infiltrate volume in lung tissue per epithelial basal membrane (b.m.) area, and (J) numbers of eosinophils on day 14 in bronchoalveolar lavage (BAL) fluid of healthy (PBS), asthmatic (HDM), or Pep19‐2.5‐treated mice (HDM + peptide), n = 10 mice per group. (K–O) Cytokine levels in BAL fluid were assessed by MSD U‐Plex assays. (P) Caspase‐1 mRNA expression in BAL cells and (Q) caspase‐1 protein determined by Western blot. (R) Final body weight on day 14 of the treatment protocol. Results are presented as mean values ± SEM. Statistical significance was assessed using ordinary one‐way ANOVA and Tukey´s multiple comparison post hoc analyses.

Article Snippet: For this purpose, Pep19‐2.5 or the specific caspase‐1 inhibitor VX765 (HY‐13205, MedChemExpress, NJ, USA) was added in triplicates to the assay buffer (pH = 7.4) consisting of 50 mM HEPES, 100 mM NaCl, 0.1% CHAPS (w/v), 1 mM EDTA, 10% glycerol (v/v), and 10 mM DTT.

Techniques: In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Membrane, Expressing, Western Blot, Comparison

Journal: iScience

Article Title: Antigen-directed single domain antibody-based TNFR1 agonists elicit preferential killing of HER2-overexpressing cancer cells

doi: 10.1016/j.isci.2026.115327

Figure Lengend Snippet: HER2-targeted TNFR1-agonists induce TNF-like caspase-1, caspase-3, and caspase-8 activation decoupled from NF-κB signaling in MCF-7 cells (A–C) Caspase-1/3/8 activation of HER2-expressing MCF-7 cells by ICM11 derivatives compared to (rh)TNF. MCF-7 cells were stimulated with increasing compound concentrations for 72 h. Caspase activities were detected intracellularly with FAM-FLICA(R) Caspase 1 Assay Kit (Biomol), CaspaTag Caspase-3 In situ Assay Kit (Merck Millipore), and CaspaTag Caspase-8 In Situ Assay Kit (Merck Millipore). Caspase-1/3/8 activation was normalized to (rh) TNF. (D) NF-κB activation in MCF-7 cells triggered by ICM11 derivatives and (rh) TNF. MCF-7 cells were stimulated with increasing concentrations of ICMs and (rh) TNF for 40 min. NF-κB was stained intracellularly with AF488-labeled anti-NF-κB staining antibody (BD) after lysis, fixation, and permeabilization of cells. (E) Remaining relative cell death of MCF-7 cells after treatment with ICM11 derivatives or (rh)TNF in the presence or absence of caspase inhibitors. MCF-7 cells were incubated with caspase-1 inhibitor (InvivoGen), caspase-3 inhibitor (R&D Systems), caspase-8 inhibitor (InvivoGen), or pan-caspase inhibitor (InvivoGen) at 50 μM and a fixed (rh) TNF or ICM concentration of 5 nM for 72 h. Killing was monitored by green fluorescence signal with SYTOX Green Dead Cell Stain and normalized to (rh) TNF signal. Mean values ±SEM of four independent experiments for each figure are shown. ∗∗∗∗ p < 0.0001,∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 calculated by utilizing two-way ANOVA multiple analyses and Bonferroni test.

Article Snippet: We also set out to investigate the consequences of caspase inhibition on cell death induction, harnessing caspase-1 inhibitor (Ac-YVAD-cmk, InvivoGen), caspase-3 inhibitor (Z-DEVD-FMK, R&D Systems), and caspase-8 inhibitor (Z-IETD-FMK, InvivoGen) as well as pan-caspase inhibitor (zVAD-FMK, InvivoGen).

Techniques: Activation Assay, Expressing, In Situ, Staining, Labeling, Lysis, Incubation, Concentration Assay, Fluorescence

Journal: Cell Death & Disease

Article Title: Lactic acid drives NLRP3 inflammasome activation and caspase-1–like cytokine cleavage via intracellular acidification

doi: 10.1038/s41419-026-08708-y

Figure Lengend Snippet: A Schematic of the experimental design. Macrophages were stimulated with nigericin or ATP in the presence of lactic acid (LA, pH 6.9), sodium lactate (NaL, pH 7.4), or hydrochloric acid (HCl, pH 6.9). IL-1β secretion and protein expression were assessed 45 min (nigericin) or 60 min (ATP) post-treatment by ELISA and western blot (WB), respectively. B IL-1β concentrations ( n = 4) and immunoblots of caspase-1 p20 and IL-1β p17 in supernatants (SN) of cells treated as indicated. The immunoblot is representative of three independent experiments. C Dose-dependent effects of LA and NaL on IL-1β secretion ( n = 6) and immunoblots of caspase-1 and IL-1β. The immunoblot is representative of three independent experiments. D IL-1β secretion ( n = 4) and immunoblots in cells treated with NaL and HCl as indicated in the presence of nigericin. The immunoblot is representative of three independent experiments. E Extracellular lactate levels measured following stimulation with nigericin ± HCl ( n = 3). F IL-1β secretion from bone marrow-derived macrophages (BMDMs) treated with LA or NaL and stimulated with nigericin (top) or ATP (bottom) ( n = 4). G IL-1β secretion in BMDMs treated with HCl and nigericin ( n = 4). All data are shown as mean ± SD. * P < 0.05, ** P < 0.01 by one-way ANOVA with Tukey’s HSD post hoc test.

Article Snippet: For pan-caspase inhibition and caspase-1-specific inhibition, THP-1-derived macrophages were treated with Z-VAD-FMK (MedChemExpress, 20 μM), or Y-VAD-FMK (MedChemExpress, 20 μM) for 1 h. For GPR81 activation or inhibition, cells were treated with 3Cl-5OH-BA (1 mM) or 3-OBA (15 mM) for 1 h. For knockdown of NLRP3 and caspase-1, THP-1-derived macrophages were transfected with siRNA specifically against NLRP3 and caspase-1 using Lipofectamine 2000 (Invitrogen) as described previously [ ].

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay

Journal: Cell Death & Disease

Article Title: Lactic acid drives NLRP3 inflammasome activation and caspase-1–like cytokine cleavage via intracellular acidification

doi: 10.1038/s41419-026-08708-y

Figure Lengend Snippet: A Extracellular lactate concentrations in THP-1-derived macrophages stimulated with nigericin ( n = 5) or ATP ( n = 3) and treated with or without the lactate dehydrogenase inhibitor GSK2837808A (GSK). * P < 0.05, ** P < 0.01, determined by one-way ANOVA with LSD post hoc test. B IL-1β secretion measured by ELISA in cells stimulated with nigericin or ATP and treated with 10 mM extracellular LA ± GSK ( n = 6). C Immunoblots of caspase-1 p20 and IL-1β p17 in supernatants from cells treated as in ( B ). Relative band intensities are shown below each blot. The immunoblot is representative of three independent experiments. D Flow cytometric analysis of ASC speck formation in ASC–mCherry-expressing THP-1 macrophages treated with nigericin and/or LA, with or without GSK. Representative plots (top) and quantification of ASC speck–positive cells (bottom); speck-positive cells were defined by reduced ASC-W:ASC-A signal profiles (boxed regions) ( n = 4). * P < 0.05, ** P < 0.01, ns, not significant by one-way ANOVA with Tukey’s HSD post hoc test. All data are shown as mean ± SD.

Article Snippet: For pan-caspase inhibition and caspase-1-specific inhibition, THP-1-derived macrophages were treated with Z-VAD-FMK (MedChemExpress, 20 μM), or Y-VAD-FMK (MedChemExpress, 20 μM) for 1 h. For GPR81 activation or inhibition, cells were treated with 3Cl-5OH-BA (1 mM) or 3-OBA (15 mM) for 1 h. For knockdown of NLRP3 and caspase-1, THP-1-derived macrophages were transfected with siRNA specifically against NLRP3 and caspase-1 using Lipofectamine 2000 (Invitrogen) as described previously [ ].

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: Lactic acid drives NLRP3 inflammasome activation and caspase-1–like cytokine cleavage via intracellular acidification

doi: 10.1038/s41419-026-08708-y

Figure Lengend Snippet: A Intracellular lactate concentrations in THP-1–derived macrophages left untreated or stimulated with nigericin ( n = 4). B Time-course analysis of mean intracellular pH (pHi) measured in live cells following nigericin stimulation ( n = 3). C Single-cell pH distribution analysis ( n = 3). D – H Flow cytometric analysis of intracellular pH using pHrodo staining in THP-1-derived macrophages. D pHrodo signal intensity in cells treated with nigericin ± GSK ( n = 3). E pHrodo intensity in cells co-treated with nigericin and LA, HCl, or NaL ( n = 4). F Effect of NaL co-treatment on HCl-induced intracellular acidification in nigericin-stimulated cells ( n = 4). G GSK abolished lactic acid–induced acidification in nigericin-stimulated cells ( n = 4). H pHrodo intensity in cells treated with NaOH (pH 8.0 or 8.5) during nigericin stimulation ( n = 3). I Immunoblots and quantification of caspase-1 p20 and IL-1β p17 in THP-1-derived macrophages treated with nigericin and increasing extracellular pH ( n = 4). The immunoblot is representative of four independent experiments. J IL-1β secretion by ELISA in THP-1-derived macrophages treated with nigericin ± NaOH. K Extracellular lactate levels measured in THP-1-derived macrophages treated with nigericin ± NaOH ( n = 4). All data are shown as mean ± SD. * P < 0.05, ** P < 0.01, NS, not significant by one-way ANOVA with Tukey’s HSD post hoc test.

Article Snippet: For pan-caspase inhibition and caspase-1-specific inhibition, THP-1-derived macrophages were treated with Z-VAD-FMK (MedChemExpress, 20 μM), or Y-VAD-FMK (MedChemExpress, 20 μM) for 1 h. For GPR81 activation or inhibition, cells were treated with 3Cl-5OH-BA (1 mM) or 3-OBA (15 mM) for 1 h. For knockdown of NLRP3 and caspase-1, THP-1-derived macrophages were transfected with siRNA specifically against NLRP3 and caspase-1 using Lipofectamine 2000 (Invitrogen) as described previously [ ].

Techniques: Derivative Assay, Single Cell, Staining, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Lactic acid drives NLRP3 inflammasome activation and caspase-1–like cytokine cleavage via intracellular acidification

doi: 10.1038/s41419-026-08708-y

Figure Lengend Snippet: A – C THP-1–derived macrophages were stimulated with nigericin in the presence or absence of extracellular lactic acid (LA, 10 mM; pH 6.9) or extracellular alkalinization (NaOH; pH 8.5). Mitochondrial membrane potential (ΔΨm) ( A ) total cellular reactive oxygen species (ROS) ( B ) and mitochondrial ROS ( C ) were assessed by TMRM ( n = 4), H₂-DCFDA ( n = 6), and MitoSOX ( n = 4) staining, respectively, followed by flow cytometric analysis. Representative histograms (top) and quantification of mean fluorescence intensity (MFI) (bottom) are shown. D Immunoblot analysis of phosphorylated PKR (p-PKR) and total PKR under the indicated conditions ( n = 4). E , F Immunoblot analysis of cleaved caspase-1 (p20) and mature IL-1β (p17) in culture supernatants ( n = 3), inflammasome components in cell lysates ( n = 4), and corresponding ELISA quantification ( n = 6) in control or PKR-silenced cells. G , H Immunoblot analysis of cleaved caspase-1 (p20) and mature IL-1β (p17) in culture supernatants ( n = 3), inflammasome components in cell lysates ( n = 4), and corresponding ELISA quantification ( n = 6) in cells treated with the PKR kinase inhibitor C16. I Immunoblot analysis of PKR and phosphorylated PKR (p-PKR) in nigericin-stimulated cells treated with the ROS scavenger N-acetylcysteine (NAC) ( n = 3). J , K Co-immunoprecipitation analysis of PKR–NLRP3 interaction in nigericin-stimulated cells treated with extracellular alkalinization ( J ) or the PKR inhibitor C16 ( K ) ( n = 3 each). All data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s HSD post hoc test or Student’s t -test, as appropriate. P < 0.05; P < 0.01; NS not significant.

Article Snippet: For pan-caspase inhibition and caspase-1-specific inhibition, THP-1-derived macrophages were treated with Z-VAD-FMK (MedChemExpress, 20 μM), or Y-VAD-FMK (MedChemExpress, 20 μM) for 1 h. For GPR81 activation or inhibition, cells were treated with 3Cl-5OH-BA (1 mM) or 3-OBA (15 mM) for 1 h. For knockdown of NLRP3 and caspase-1, THP-1-derived macrophages were transfected with siRNA specifically against NLRP3 and caspase-1 using Lipofectamine 2000 (Invitrogen) as described previously [ ].

Techniques: Derivative Assay, Membrane, Staining, Fluorescence, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: Lactic acid drives NLRP3 inflammasome activation and caspase-1–like cytokine cleavage via intracellular acidification

doi: 10.1038/s41419-026-08708-y

Figure Lengend Snippet: A Schematic of the cell-free NLRP3 inflammasome activation assay. Whole-cell lysates (CL) from THP-1-derived macrophages or HEK293T cells were incubated with LA, NaL, or HCl, followed by immunoblot analysis. B Dose-dependent effects of LA on IL-1β cleavage in macrophage lysates. Immunoblots show IL-1β, pro-IL-1β, caspase-1, and procaspase-1. Densitometric quantification of IL-1β relative to pro-IL-1β is shown (bottom, n = 6). PC, positive control (nigericin-treated lysates). Immunoblots of IL-1β in lysates from cells with siRNA knockdown of NLRP3 ( C ) ASC ( D ) or caspase-1 ( E ) followed by LA treatment. GAPDH and pro-IL-1β serve as loading controls. The immunoblot is representative of three independent experiments. F Immunoblots of IL-1β in macrophage lysates treated with LA in the presence of the pan-caspase inhibitor Z-VAD-FMK or the caspase-1–specific inhibitor Y-VAD-FMK. The immunoblot is representative of three independent experiments. G Immunoblots of IL-1β in HEK293T lysates transfected with pro-IL-1β or empty vector (EV), followed by LA treatment. The immunoblot is representative of three independent experiments. H Immunoblot of IL-1β and pro-IL-1β in macrophage lysates treated with LA. Densitometric quantification of IL-1β relative to pro-IL-1β is shown (bottom, n = 3). All data are shown as mean ± SD. * P < 0.05, ** P < 0.01, NS, not significant by one-way ANOVA with Tukey’s HSD post hoc test.

Article Snippet: For pan-caspase inhibition and caspase-1-specific inhibition, THP-1-derived macrophages were treated with Z-VAD-FMK (MedChemExpress, 20 μM), or Y-VAD-FMK (MedChemExpress, 20 μM) for 1 h. For GPR81 activation or inhibition, cells were treated with 3Cl-5OH-BA (1 mM) or 3-OBA (15 mM) for 1 h. For knockdown of NLRP3 and caspase-1, THP-1-derived macrophages were transfected with siRNA specifically against NLRP3 and caspase-1 using Lipofectamine 2000 (Invitrogen) as described previously [ ].

Techniques: Activation Assay, Derivative Assay, Incubation, Western Blot, Positive Control, Knockdown, Transfection, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Lactic acid drives NLRP3 inflammasome activation and caspase-1–like cytokine cleavage via intracellular acidification

doi: 10.1038/s41419-026-08708-y

Figure Lengend Snippet: A Immunoblot of IL-1β–FLAG generated from immunoprecipitated pro-IL-1β–FLAG protein using anti-FLAG antibody from HEK293T cells transfected with pro-IL-1β–FLAG, followed by treatment with LA. The immunoblot is representative of three independent experiments. B Immunoblot of IL-18 generated from recombinant pro-IL-18 protein treated with LA or active caspase-1 (positive control). The immunoblot is representative of three independent experiments. C Recombinant pro-IL-1β protein was incubated with NaL across a pH gradient or with active caspase-1. Top: immunoblot showing pH-dependent cleavage of pro-IL-1β into IL-1β. Bottom: graph showing correlation between pH-dependent conversion of lactate to LA (calculated using the Henderson–Hasselbalch equation) and IL-1β generation ( n = 3). D Recombinant pro-IL-1β was treated with LA, NaL, HCl, or acetic acid (AcOH). Top: immunoblot showing IL-1β p17 production; bottom: densitometric quantification of IL-1β p17 expression ( n = 4). All data are shown as mean ± SD. * P < 0.05, ** P < 0.01, NS, not significant by one-way ANOVA with Tukey’s HSD post hoc test.

Article Snippet: For pan-caspase inhibition and caspase-1-specific inhibition, THP-1-derived macrophages were treated with Z-VAD-FMK (MedChemExpress, 20 μM), or Y-VAD-FMK (MedChemExpress, 20 μM) for 1 h. For GPR81 activation or inhibition, cells were treated with 3Cl-5OH-BA (1 mM) or 3-OBA (15 mM) for 1 h. For knockdown of NLRP3 and caspase-1, THP-1-derived macrophages were transfected with siRNA specifically against NLRP3 and caspase-1 using Lipofectamine 2000 (Invitrogen) as described previously [ ].

Techniques: Western Blot, Generated, Immunoprecipitation, Transfection, Recombinant, Positive Control, Incubation, Expressing

Journal: Cell Death & Disease

Article Title: Lactic acid drives NLRP3 inflammasome activation and caspase-1–like cytokine cleavage via intracellular acidification

doi: 10.1038/s41419-026-08708-y

Figure Lengend Snippet: A Schematic of the workflow for identifying the N-terminal sequence of mature-like IL-1β using dimethyl labeling and LC-MS/MS following SDS-PAGE, in-gel digestion, and chymotrypsin cleavage. B Coomassie staining (top) and immunoblotting (bottom) of IL-1β in samples treated with LA or active caspase-1. Bands corresponding to cleaved IL-1β were excised for mass spectrometry analysis. The immunoblot is representative of three independent experiments. C Representative MS/MS spectra showing the identical N-terminal sequence (APVRSLNCTL) of IL-1β generated by LA or caspase-1 cleavage. Schematic (bottom) shows the cleavage site at Asp116 in the context of full-length IL-1β. D Immunoblot of IL-1β-FLAG cleavage in immunoprecipitated lysates from 293T cells expressing wild-type (WT) or Asp116 mutants (D116A, D116N) of pro-IL-1β-FLAG, treated with LA. The immunoblot is representative of three independent experiments.

Article Snippet: For pan-caspase inhibition and caspase-1-specific inhibition, THP-1-derived macrophages were treated with Z-VAD-FMK (MedChemExpress, 20 μM), or Y-VAD-FMK (MedChemExpress, 20 μM) for 1 h. For GPR81 activation or inhibition, cells were treated with 3Cl-5OH-BA (1 mM) or 3-OBA (15 mM) for 1 h. For knockdown of NLRP3 and caspase-1, THP-1-derived macrophages were transfected with siRNA specifically against NLRP3 and caspase-1 using Lipofectamine 2000 (Invitrogen) as described previously [ ].

Techniques: Sequencing, Labeling, Liquid Chromatography with Mass Spectroscopy, SDS Page, Staining, Western Blot, Mass Spectrometry, Tandem Mass Spectroscopy, Generated, Immunoprecipitation, Expressing

Journal: Journal of Advanced Research

Article Title: Designing an apoptosis reporter by mutagenesis-based insertion of caspase-3 cleavage motif into green fluorescence protein

doi: 10.1016/j.jare.2025.06.070

Figure Lengend Snippet: Monitoring of apoptosis reporter in live cells. (A) HEK293 EGFP #4-8 cells were cultured with or without 1 μM STA, and images were obtained every 30 min for 48 h; scale bar 50 μm. Relative EGFP fluorescence intensity was measured using ImageJ software (N = 3). (B) Protein levels of GFP, caspase-3, cleaved caspase-3, cytochrome C , PARP, and GAPDH by Western blotting in HEK293 EGFP #4-8 cells incubated with STA. Graph, protein levels (N = 3). (C) HEK293 EGFP #4-8 cells were cultured with 1 μM STA co-treated with or without 20 μM Z-DEVD-FMK and 10 μM Z-YVAD-FMK, and images were obtained every 30 min for 48 h. Relative EGFP fluorescence intensity was measured using ImageJ software (N = 3). (D) HEK293 EGFP #4-8 cells were cultured with 100, 200, and 500 μM H 2 O 2 , and images were obtained every 30 min for 24 h. Relative EGFP fluorescence intensity was measured using ImageJ software (N = 3). Error bars are ± SD. *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001.

Article Snippet: Z-DEVD-FMK (caspase-3 inhibitor, #sc-311558) and Z-YVAD-FMK (caspase-1 inhibitor, #sc-3071) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Cell Culture, Fluorescence, Software, Western Blot, Incubation