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