Journal: Cell reports

Article Title: Structural homology screens reveal host-derived poxvirus protein families impacting inflammasome activity

doi: 10.1016/j.celrep.2023.112878

Figure Lengend Snippet: (A) A crystal structure of Eptesipox virus (EPTV) gasdermin reveals homology to mammalian gasdermins. Top: domain organization of mouse GSDMA3 (mGSDMA3) and EPTV gasdermin indicating the pore-forming N-terminal domain (NTD) and the autoinhibitory C-terminal domain (CTD). Middle: crystal structures of full-length mGSDMA3 (PDB: 5B5R) and EPTV gasdermin (this study). The N and C termini are indicated with the circled letters N and C, and the dashed box and oval indicate the first α-helix of each structure. Bottom left: superposition of mGSDMA3 and EPTV gasdermin predicts a steric clash between the α1 helices of each protein. Bottom right: topology diagrams of the mGSDMA3 CTD and EPTV gasdermin indicate a conserved region of gasdermin homology. (B) Overview of probable gasdermin gain/loss events based on phylogenetic analyses in , , and . Gasdermins were identified by a combination of sequence and structure-based approaches; see for details. (C) Maximum likelihood tree of select vertebrate gasdermin regulatory domains and poxvirus gasdermins. Clusters of vertebrate gasdermins are labeled based on human gasdermins. Bootstrap values from 1,000 replicates are indicated for select branches. Scale: AA substitutions per site. (D) Top: HeLa cells expressing GSDMD-CTD, vaccina GSDM, or a vector control were electroporated with LPS to activate the noncanonical inflammasome. Viability as assessed by ATP levels relative to mock are indicated. n = 3 biological replicates. One-way ANOVA with Tukey’s multiple comparison test. Data are represented as mean ± SD. Bottom: alongside one replicate, levels of transfected gasdermin proteins in mock-electroporated wells were assessed by western blotting. (E) The murine NLRP3 inflammasome was reconstituted in 293T cells stably expressing A47L or a vector control. Following nigericin stimulation, IL-1β concentrations in supernatants were quantified by ELISA. n = 3 biological replicates. Two-way ANOVA with Šídák’s multiple comparisons test. Data are represented as mean ± SD. (F) Vaccinia replication (inoculum MOI of 1) in LPS-primed murine macrophages was measured by TCID50. n = 3 biological replicates. Two-way ANOVA with Fisher’s least significant difference (LSD) test. Data are represented as mean ± SD. ANOVA statistic for viral strain across time points: p = 0.0118. (G) LPS-primed murine macrophages were infected with wild-type (VC2) or A47-deficient vaccinia virus at an MOI of 1, and IL-1β was quantified by ELISA 1 day post-infection. n = 3 biological replicates. Paired t test. Data are represented as mean ± SD.

Article Snippet: Nigericin sodium salt , ThermoFisher , J61349.MA.

Techniques: Virus, Sequencing, Labeling, Expressing, Plasmid Preparation, Control, Comparison, Transfection, Western Blot, Stable Transfection, Enzyme-linked Immunosorbent Assay, Infection

Journal: Cell reports

Article Title: Structural homology screens reveal host-derived poxvirus protein families impacting inflammasome activity

doi: 10.1016/j.celrep.2023.112878

Figure Lengend Snippet: (A) Top: 293T cells were co-transfected with the indicated plasmids, a plasmid encoding firefly luciferase driven by an NF-κB promoter, and a constitutive Renilla luciferase plasmid as a transfection control. 24 h post-transfection, cells were treated with 1 ng/mL TNF-α, and promoter activity was assessed by luciferase assay 6 h post-transfection. n = 3 biological replicates. Repeated measures (RM) one-way ANOVA with Dunnett’s correction. Data are represented as mean ± SD. Bottom: in parallel, cells were transfected with equal amounts of the indicated plasmids, and protein expression was determined by western blotting. (B) 293T cells were co-transfected with a plasmid encoding FLAG-C1 and either ASC-GFP or an empty vector. 24 h post-transfection, cells were fixed and stained for FLAG (C1) and subsequently imaged. Representative image from n = 3 independent experiments. Scale bar: 5 μm. See also and . (C) 293T cells were co-transfected with plasmids encoding the NLRP3 inflammasome components NLRP3, ASC, NEK7, caspase-1, and pro-IL-1β, in addition to either C1 or a GFP control. Following nigericin stimulation, IL-1β concentrations in supernatants were quantified by ELISA. n = 3 biological replicates. Two-way ANOVA with Šídák’s multiple comparisons test. Data are represented as mean ± SD. (D) 293T cells were co-transfected as in (C) but with NLRP3 replaced by an empty vector. Cells were not stimulated with nigericin. IL-1β concentrations in supernatants were quantified by ELISA. n = 3 biological replicates. Paired t test. Data are represented as mean ± SD. (E) 293T cells were co-transfected as in (C) but with ASC replaced by an empty vector where indicated. Cells were not stimulated with nigericin. IL-1β concentrations in supernatants were quantified by ELISA. n = 3 biological replicates. Two-way ANOVA with Šídák’s multiple comparisons test. Data are represented as mean ± SD. (F) Vaccinia replication (inoculum MOI of 1) in LPS-primed murine macrophages was measured by TCID50. Data are represented as mean ± SD. (G) LPS-primed murine macrophages were infected with wild-type (VC2) or C1L-deficient vaccinia virus at an MOI of 10, and IL-1β was quantified by ELISA 4 h post-infection. n = 3 biological replicates. Paired t test. Data are represented as mean ± SD.

Article Snippet: Nigericin sodium salt , ThermoFisher , J61349.MA.

Techniques: Transfection, Plasmid Preparation, Luciferase, Control, Activity Assay, Expressing, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Infection, Virus

Journal: Cell reports

Article Title: Structural homology screens reveal host-derived poxvirus protein families impacting inflammasome activity

doi: 10.1016/j.celrep.2023.112878

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Nigericin sodium salt , ThermoFisher , J61349.MA.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Ligation, Sequencing, Software

Journal: Mediators of Inflammation

Article Title: GPR34 Stabilized by Deubiquitinase USP8 Suppresses Ferroptosis of ATC

doi: 10.1155/mi/5576056

Figure Lengend Snippet: GPR34 deletion promotes ferroptosis of ATC cells. (A, B) The CCK8 assay was used to determine the viability of BHT101 (A) and 8305C (B) cells with GPR34 knockdown after addition of the apoptosis inducer VCR, ferroptosis inducer RSL-3, autophagy inducer rapamycin, or pyroptosis inducer LPS + nigericin. (C, D) Representative images were presented of BHT101 (C) and 8305C cells (D) with GPR34 deletion treated with RSL-3. (E, F) The CCK8 assay was used to assess the responses of BHT101 (E) and 8305C cells (F) with GPR34 silencing to RSL-3 ± Fer-1. (G) IHC test on xenograft tumors was performed to identify the correlation between GPR34 and 4-HNE expression. (H, I) Lipid ROS (H) and GSH (I) levels in GPR34 deletion ATC cells. Ns, nonsignificant. p > 0.05, ⁣ ∗ p < 0.05, ⁣ ∗∗ p < 0.01, ⁣ ∗∗∗ p < 0.001.

Article Snippet: Apoptosis inducer VCR (S1241), autophagy inducer Rapamycin (S1039), and pyroptosis inducer LPS (S7850) + Nigericin (S6653) were provided by Selleck.

Techniques: CCK-8 Assay, Knockdown, Expressing

Journal: Inflammopharmacology

Article Title: Dexketoprofen enhances NLRP3 activation via ATPase activity after canonical stimuli

doi: 10.1007/s10787-025-01928-2

Figure Lengend Snippet: Effect of DXK alone and with inflammatory stimulation in human macrophages. A Representative images of western blot analysis of macrophages exposed to DXK 100 nM for 15 min and 1 nM, primed with LPS 1 μg/mL for 4 h stimulated with nigericin 10 µM for 30 min. B IL-1β concentration in the supernatant measured by ELISA. C LDH activity measured in the supernatant. D DeltaVision confocal images of human macrophages exposed to DXK 100 nM and 1 nM, primed with LPS and stimulated with nigericin. Macrophages were labeled with ASC (green) and DAPI (blue). Scale bars, 10 µm. Data are mean + SEM from n = 3 independent experiments. E The percentage of ASC speck-containing cells was quantified using the imaging software Fiji. For each individual experiment, the average number of ASC specks per nucleus was calculated and plotted. A total of 100 cells per condition were imaged and quantified. Data are mean + SEM from n = 3 independent experiments. Data were analyzed using a Student’s t test. ns = p > 0.05, *, a = p ≤ 0.05, **, aa = p ≤ 0.01. * represents treatment vs control and a represents treatment vs LPS-Nig

Article Snippet: Phorbol 12-myristate-13-acetate (PMA) (P8139-MG, Sigma Chemical Co., St. Louis, MO, USA), dexketoprofen (sc-357330, Santa Cruz Biotechnology, Santa Cruz, CA, USA), lipopolysaccharide (LPS) (L4391, Sigma Chemical Co., St. Louis, MO, USA), nigericin (sc-201518B, Santa Cruz, CA, USA), adenosine triphosphate (ATP) (sc-202040A, Santa Cruz, CA, USA), bovine serum albumin (BSA) (9048–46-8, Sigma Chemical Co., St. Louis, MO, USA), DAPI (Santa Cruz Biotechnology, Santa Cruz, CA, USA), saponin (47,036, Sigma Chemical Co., St. Louis, MO, USA), bioBLUPrestainesProtein loader (GTPBM0003, Biorad Laboratories Inc., Hercules, CA, USA).

Techniques: Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Labeling, Imaging, Software, Control

Journal: Inflammopharmacology

Article Title: Dexketoprofen enhances NLRP3 activation via ATPase activity after canonical stimuli

doi: 10.1007/s10787-025-01928-2

Figure Lengend Snippet: Combined effect of DXK and MCC950 in human macrophages when exposed to nigericin. A DXK docked to the NATCH domain of human NLRP3 (PDB:6NPY) using AutoDock Vina . B Chemical structure of DXK and zoom-in of the binding pocket with a score of -8.4 kcal/mol. Hydrogen bonds in blue. C ATPase activity measured by ADP-Glo Kinase Assay Kit using purified human NLRP3 exposed 15 min to vehicle, DXK 10 µM, MCC950 10 µM and both before adding purified ATP 250 µM. Data of enzyme activity were normalized to vehicle. D IL-1β concentration in the supernatant from macrophages exposed 15 min to DXK 100 nM, MCC950 10 µM and both before nigericin 10 µM 45 min. E LDH activity measured in the supernatant. Data are mean + SEM from n = 3 independent experiments. Data were analyzed using a Student’s t test. ns = p > 0.05, *, a = p ≤ 0.05, **, aa = p ≤ 0.01. * represents treatment vs control and a represents treatment vs Nig in C and D. * represents DXK vs control and a represents MCC950 vs DXK and vs Nig in E

Article Snippet: Phorbol 12-myristate-13-acetate (PMA) (P8139-MG, Sigma Chemical Co., St. Louis, MO, USA), dexketoprofen (sc-357330, Santa Cruz Biotechnology, Santa Cruz, CA, USA), lipopolysaccharide (LPS) (L4391, Sigma Chemical Co., St. Louis, MO, USA), nigericin (sc-201518B, Santa Cruz, CA, USA), adenosine triphosphate (ATP) (sc-202040A, Santa Cruz, CA, USA), bovine serum albumin (BSA) (9048–46-8, Sigma Chemical Co., St. Louis, MO, USA), DAPI (Santa Cruz Biotechnology, Santa Cruz, CA, USA), saponin (47,036, Sigma Chemical Co., St. Louis, MO, USA), bioBLUPrestainesProtein loader (GTPBM0003, Biorad Laboratories Inc., Hercules, CA, USA).

Techniques: Binding Assay, Activity Assay, Kinase Assay, Purification, Concentration Assay, Control

Journal: Frontiers in Immunology

Article Title: Exposure to Dengue Envelope Protein Domain III Induces Nlrp3 Inflammasome-Dependent Endothelial Dysfunction and Hemorrhage in Mice

doi: 10.3389/fimmu.2021.617251

Figure Lengend Snippet: DENV- and rEIII-induced RCD in endothelial cells. (A) HMEC-1 endothelial cells treated with vehicle and various doses of DENV and rEIII; the live or dead status of the cell populations were determined using Zombie-NIR Kit labeling and flow cytometry analysis. [rEIII 1× = 0.3 µM, 2× = 0.6 µM, 4× = 1.2 µM; DENV e(rEIII 1×) is a DENV level equivalent to 0.3 µM rEIII]. (B) RCD inducers, doxorubicin (DOX; apoptosis) (2.5 µg/ml), rapamycin (autophagy) (0.5 µM), erastin (ferroptosis) (10 µM), TNF-α (necroptosis) (2.5 ng/ml), and nigericin (pyroptosis) (3.5 µM) induced relatively simple RCD patterns (flow cytometry gating and calculation methods described in Figure S3 ). By contrast, DENV and rEIII induced multiple RCD pathways, in which pyroptosis was the main RCD response, accounting for approximately 50% of total RCD response. If we normalize the respective RCD by the population of death cells (dead cell population normalized to 100%), we can obtain a more similar RCD pattern in DENV and rEIII groups (C) . ** P < 0.01 vs. vehicle groups.

Article Snippet: Treatments (1 h) of cell death inducers were used as positive controls for various types of RCD (apoptosis: doxorubicin, 2.5 μg/ml, Nang Kuang Pharmaceutical, Taipei, Taiwan; autophagy: rapamycin, 250 nM, Sigma-Aldrich; ferroptosis: erastin, 10 μM, Cayman Chemical; necroptosis: tumor necrosis factor alpha [TNF-α], 2 ng/ml, BioLegend; pyroptosis: nigericin, 3.5 μM, ImmunoChemistry Technologies, Minnesota, USA; 30 min in PBS).

Techniques: Labeling, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Exposure to Dengue Envelope Protein Domain III Induces Nlrp3 Inflammasome-Dependent Endothelial Dysfunction and Hemorrhage in Mice

doi: 10.3389/fimmu.2021.617251

Figure Lengend Snippet: Protection of endothelial cells from rEIII-induced pyroptosis under treatment with Nlrp3 inflammasome inhibitors. Treatment with Nlrp3 inhibitor CSB (chondroitin sulfate B, 10 μg/ml), OLT1177 (10 µM) and caspase 1 inhibitor Z-WHED-FMK (10 µM) rescued rEIII-induced endothelial cell death (A) . Treatments with CSB, OLT1177 and Z-WHED-FMK rescued cell dead population ( B, C, F, G, J, K ; cells with RCDs). If we normalize the respective RCD % by the population of death cells ( D : dead cell population normalized to 100%), we found that CSB, OLT1177 and Z-WHED-FMK preferentially rescued pyroptosis (E) , but not the other tested RCDs (H, I, L, M) . n = 6, ** P < 0.01, significant suppression vs. vehicle groups.

Article Snippet: Treatments (1 h) of cell death inducers were used as positive controls for various types of RCD (apoptosis: doxorubicin, 2.5 μg/ml, Nang Kuang Pharmaceutical, Taipei, Taiwan; autophagy: rapamycin, 250 nM, Sigma-Aldrich; ferroptosis: erastin, 10 μM, Cayman Chemical; necroptosis: tumor necrosis factor alpha [TNF-α], 2 ng/ml, BioLegend; pyroptosis: nigericin, 3.5 μM, ImmunoChemistry Technologies, Minnesota, USA; 30 min in PBS).

Techniques: