Journal: Emerging Microbes & Infections

Article Title: Vibrio pore-forming leukocidin activates pyroptotic cell death via the NLRP3 inflammasome

doi: 10.1080/22221751.2020.1720526

Figure Lengend Snippet: Inflammasome inhibitors delay VPRH-mediated cell death. Approximately 3.5*10 4 wild-type BMDMs were seeded into 96-well plates in triplicate, and were primed using LPS (100 ng/ml) for 3 h prior to infection with V. proteolyticus mutants at MOI 50. Where indicated, inflammasome inhibitors – Vx765 (25 μM), MCC950 (20 μM), or NSA (20 μM), with the addition of propidium iodide (PI) (1 μg/ml), were added to the cells 30 min prior to bacterial infection. DMSO was added as the solvent control. (A) PI uptake was assessed using real-time microscopy (IncucyteZOOM) and then graphed as the percentage of PI-positive cells normalized to the number of cells in wells. Data are presented as the mean ± SD; n = 3. (B) Summary of normalized AUC for three independent biological experiments shown in panel A. (C-E) Cell lysates and supernatants from experiments described in A were collected 6 h post-infection. (C, D) IL-1β (C) and TNFα (D) secretion were measured using commercial ELISA kits. (E, F) Inflammasome components, caspase-1 (Casp1), GSDMD, and IL-1β cleavage were detected in BMDM lysates (E) and supernatants (F) using immunoblots (the numbers on the right of each blot indicate the blot number). The data in A, E, and F are representative of 3 independent experiments. Statistical comparisons in B, C and D between the different bacterial mutants and inflammasome inhibitors were carried using RM two-way ANOVA, followed by Tukey's multiple comparison test; the results are presented as the mean (bars) ± SD of 3 independent experiments; significant differences are denoted only for comparisons between inhibitors in each infected strain; * P < 0.05, ** P < 0.01.

Article Snippet: Necrosulfinamide (NSA) was purchased from Tocris Bioscience; Vx765 and MCC950 were purchased from Invitrogen.

Techniques: Infection, Solvent, Control, Microscopy, Enzyme-linked Immunosorbent Assay, Western Blot, Comparison

Journal: Emerging Microbes & Infections

Article Title: Vibrio pore-forming leukocidin activates pyroptotic cell death via the NLRP3 inflammasome

doi: 10.1080/22221751.2020.1720526

Figure Lengend Snippet: Inflammasome inhibitors delay VPRH-mediated cell death in human PBMCs. Approximately 2*10 4 healthy donor PBMCs were seeded into 96-well plates in triplicate for 18 h prior to infection with V. proteolyticus mutants at MOI 50. Where indicated, inflammasome inhibitors – Vx765 (25 μM) or MCC950 (20 μM), with the addition of propidium iodide (PI) (1 μg/ml), were added to the cells 30 min prior to bacterial infection. DMSO was added as the solvent control. In donor 1, only the inhibitor VX765 was applied. (A) PI uptake was assessed using real-time microscopy (IncucyteZOOM) and then graphed as the percentage of PI-positive cells normalized to the number of cells in wells. Data are presented as the mean ± SD; n = 3. (B) Summary of normalized AUC for the three donors shown in panel A. (C) Supernatants from experiments described in A were collected 4 h post-infection, and IL-1β secretion was measured using commercial ELISA kit. Statistical comparisons in B and C between the different bacterial mutants and inflammasome inhibitors were carried using RM two-way ANOVA, followed by Tukey's multiple comparison test; the results are presented as the mean (bars) ± SD of 3 independent experiments; significant differences are denoted only for the comparisons between PBMCs infected with WT V. proteolyticus and the other condition; ** P < 0.01.

Article Snippet: Necrosulfinamide (NSA) was purchased from Tocris Bioscience; Vx765 and MCC950 were purchased from Invitrogen.

Techniques: Infection, Solvent, Control, Microscopy, Enzyme-linked Immunosorbent Assay, Comparison

Journal: bioRxiv

Article Title: NLRP3 acts as a direct sensor of intracellular potassium ions

doi: 10.64898/2026.03.12.707678

Figure Lengend Snippet: (A) Immunoblot of NLRP3-mNG-expressing and nigericin-treated HEK293T cell lysates prepared by sonication in K⁺ (150 mM KCl) or Na⁺ (150 mM NaCl) buffer, followed by ± MCC950 and subsequent pronase addition (undigested lysates shown in Fig. S5A). Anti-NLRP3 antibodies were used. Data are representative of n=4 independent experiments. (B) Quantification of A combining n=4 independent experiments. (C) Immunoblot of LPS-primed, VX-765 pre-treated, and nigericin-treated regular THP-1 cell lysates prepared by sonication in K⁺ (150 mM KCl) or Na⁺ (150 mM NaCl) buffer, followed by ± MCC950 and subsequent pronase addition (undigested lysates shown in Fig. S5B). Anti-NLRP3 antibodies were used. Data are representative of n=2 independent experiments. (D) Quantification of C showing combined data from n=2 independent experiments. (E) As in C but with CL097 instead of nigericin treatment and addition of 40 mM KCl addition to the media (undigested lysates shown in Fig. S5C). Data are representative of n=2 independent experiments. (F) Quantification of E showing combined data from n=2 independent experiments. (G) Immunoblot of cell lysates from NLRP3 WT-mNG or NLRP3 T350M-mNG-texpressing HEK293T cells with concomitant MCC950 treatment. Cell lysates were prepared by sonication in K⁺ (150 mM KCl) or Na⁺ (150 mM NaCl) buffer including DMSO or MCC950, followed by pronase addition (quantification in Fig. S5D, undigested lysates shown in Fig. S5E). Anti-NLRP3 antibodies were used. Data are representative of n=3 independent experiments. (H) Immunoblot of LPS-primed NLRP3 WT-mNG or NLRP3 T350M-mNG reconstituted THP-1 NLRP3 KO cell lysates prepared by sonication in K⁺ (150 mM KCl) or Na⁺ (150 mM NaCl) buffer, followed by ± MCC950 for 72 hours and subsequent pronase addition (undigested lysates shown in Fig. S5H). Anti-NLRP3 antibodies were used. Data are representative of n=2 independent experiments. (I) Immunoblot of healthy donor or CAPS T350 patient PBMC prepared by sonication in K⁺ (150 mM KCl) or Na⁺ (150 mM NaCl) buffer, followed by ± MCC950 and pronase addition (undigested lysates shown in Fig. S5I). Anti-NLRP3 antibodies were used. n=1 independent experiment with samples from 1 healthy donor and 1 CAPS patient. (J) Structure of NLRP3 face-to-face (F2F) dimers extracted from the 7PPZC decameric cage and overlaid with K + computer densities (magenta) calculated from MDS. Selected residues mutated in CAPS are highlighted as red spheres. Data are representative from n=8 independent MDS ( cf . ).

Article Snippet: Phorbol 12-myristate 13-acetate (PMA, tlrl-pma), LPS ultrapure from Escherichia coli K12 (LPS-EK, tlr-peklps), VX-765 (inh-vx765i-1), nigericin sodium salt (tlr-nig) and CL097 (tlrl-c97) were obtained from InvivoGen.

Techniques: Western Blot, Expressing, Sonication

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: Infection, Staining, Fluorescence, Microscopy

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: bioRxiv

Article Title: GSDMD pore formation regulates caspase-4 cleavage to limit IL-18 production in the intestinal epithelium

doi: 10.1101/2024.02.01.578487

Figure Lengend Snippet: HCT116 epithelial cells were treated with 2µM of pan-caspase inhibitor Z-VAD-FMK (a-c), siRNA targeting caspase-4 (d,e) or with caspase-1/4 specific inhibitor VX-765 (j-l). C2bbe1 epithelial cell double caspase-1 or 5 and GSDMD knockdowns were generated as indicated (f-i). For inflammasome activation, cells were electroporated with 2µg LPS. Cell death was measured by LDH cytotoxicity assay(a). Western blots to assess inflammasome activation were conducted on whole protein lysates (d, f, h, j). IL-18 release was measured by ELISA on supernatant alone (b, k). Total IL-18 production was measured by lysing cells into supernatant prior to ELISA (c, e, g, i, l). Representative of at least 3 experiments, data displayed as mean ± S.E.M. ****p<0.001, ***p<0.005.

Article Snippet: Rabbit anti-GSDMD (HPA044487; Sigma), mouse anti - Caspase-4 (M029-3, Marine BL), rabbit anti-caspase-1 (3866S; Cell Signalling Technology), rabbit anti-Caspase-5 (ab40887; abcam), goat anti-IL-18 (AF2548; Novus Biologicals), LPS (14011S; CST), human IFNγ (80385S; Cell Signalling Technology), Nigericin (N7143; Sigma), Flagellin (tlrl-epstfla; InvivoGen), Z-VAD-FMK (tlrl-vad; InvivoGen), VX-765 (7143/50, Cedarlane), peroxidase conjugated anti-rabbit and anti-mouse secondaries (Jackson Labs, 111-035-003), peroxidase conjugated anti-goat secondary (HAF109; R&D Systems).

Techniques: Generated, Activation Assay, LDH Cytotoxicity Assay, Western Blot, Enzyme-linked Immunosorbent Assay