Journal: Communications Biology

Article Title: Activation of the NLRP1B inflammasome by caspase-8

doi: 10.1038/s42003-024-06882-3

Figure Lengend Snippet: a RAW264.7 cells were treated with LPS and Actinomycin D (ActD) for 2 h and analyzed for Il1b transcript levels using quantitative real-time PCR. b RAW264.7 cells were treated with LPS, 2DG and ActD for 2 h. Cell lysates were immunoblotted for caspase-1, caspase-8 and β-actin. c , d RAW264.7 cells (wild-type, Tlr4 –/– , Myd88 –/– , Ticam1 –/– ) were treated with LPS/2DG for 3 h ( c ) or 2 h ( d ) ( Ticam1 encodes TRIF). Cells were stained with propidium iodide (PI) and analyzed by flow cytometry; or cell lysates were immunoblotted for caspase-1, caspase-8, and β-actin. e RAW264.7 cells were treated with LPS, 2DG and dynasore for 2 h. Cell lysates were immunoblotted for caspase-1, caspase-8 and β-actin. f Bone marrow-derived macrophages (BMDMs) were treated with LPS, 2DG and dynasore for 2 h. Cell lysates were immunoblotted for caspase-1, caspase-8 and β-actin. Blots are representative of three independent experiments. Error bars represent standard error of the mean of three independent experiments. Statistical significance was determined by a one-way ( a ) or two-way ANOVA followed by a Tukey’s post hoc test. * p < 0.05; **** p < 0.0001; NS nonsignificant.

Article Snippet: Cells were incubated at 4 ˚C for 20 min with 1 μL mouse Fc block™ (BD Biosciences, #553141; RRID:AB_394656), then incubated in the dark at 4 ˚C for 30 min with 2.5 μL APC-conjugated TLR4/MD2 antibody (Thermo Fisher, #17-9924-82; RRID:AB_657858).

Techniques: Real-time Polymerase Chain Reaction, Staining, Flow Cytometry, Derivative Assay

Journal: Communications Biology

Article Title: Activation of the NLRP1B inflammasome by caspase-8

doi: 10.1038/s42003-024-06882-3

Figure Lengend Snippet: a RAW264.7 cells (wild-type or Casp8 –/– ) were treated for 1 h with LPS and 2DG. Endogenous RIPK1 was immunoprecipitated from cell lysates with an anti-RIPK1 antibody and then immunoblotted for pro-caspase-8 and RIPK1. Mouse IgG1 was used as a control. b , c RAW264.7 cells (wild-type or Ticam -/- ) were treated for 30 min with LPS, 2DG and the TAK1 inhibitor 5z-7-Oxozeaenol (5z-7) as indicated. Endogenous RIPK1 was immunoprecipitated from cell lysates and immunoblotted for TLR4 and RIPK1. d RAW264.7 cells were treated for 30 min with LPS, 2DG and 5z-7 and cell lysates were immunoblotted for RIPK1. e RAW264.7 cells were treated for 30 min with LPS. Cell lysates were incubated in Alkaline Phosphatase (AP) for 30 min and then immunoblotted for RIPK1. f RAW264.7 cells were treated for 2 h with LPS, 2DG and 5z-7. Cell lysates were immunoblotted for pro-caspase-8. g C57BL/6 BMDMs were treated as in ( d ). h RAW264.7 and ( i ) C57BL/6 BMDMs were treated with LPS, 2DG, for 2 h and TNFα/SMAC mimetic/Z-VAD (T/S/Z) for 3 h and immunoblotted for pro-caspase-8, phospho-MLKL S345, MLKL, and β-actin. Blots are representative of three independent experiments.

Article Snippet: Cells were incubated at 4 ˚C for 20 min with 1 μL mouse Fc block™ (BD Biosciences, #553141; RRID:AB_394656), then incubated in the dark at 4 ˚C for 30 min with 2.5 μL APC-conjugated TLR4/MD2 antibody (Thermo Fisher, #17-9924-82; RRID:AB_657858).

Techniques: Immunoprecipitation, Control, Incubation

Journal: Communications Biology

Article Title: Activation of the NLRP1B inflammasome by caspase-8

doi: 10.1038/s42003-024-06882-3

Figure Lengend Snippet: a RAW264.7 cells were incubated with Shigella flexneri at indicated multiplicities of infection (MOI) for 1 h. Cell lysates were immunoblotted for caspase-1, pro-caspase-8, cleaved caspase-8, and β-actin. b , c Shigella flexneri (wild-type, M90T; and mutant, BS176) were incubated with RAW264.7 cells at an MOI of 5 for 1 h. Intracellular ATP concentrations were measured and cell lysates were immunoblotted for caspase-1, pro-caspase-8, cleaved caspase-8 and β-actin. d RAW264.7 cells (wild-type, Nlrp1b –/– , Tlr4 –/– , Casp8 –/– ) were incubated with Shigella flexneri at an MOI of 5 for 1 h. Cell lysates were immunoblotted for caspase-1, pro-caspase-8, cleaved caspase-8, and β-actin. e , f RAW264.7 cells were incubated with lethal toxin (LT) for 3 h, Shigella flexneri for 1 h, LPS/2DG, and MG132 for 2 h. Cell lysates were immunoblotted for caspase-1, GSDMD, and β-actin. Blots are representative of at least three independent experiments. Error bars represent standard error of the mean of three independent experiments. Statistical significance was determined using a one-way ANOVA followed by a Tukey’s post hoc test. *** p < 0.001; NS nonsignificant.

Article Snippet: Cells were incubated at 4 ˚C for 20 min with 1 μL mouse Fc block™ (BD Biosciences, #553141; RRID:AB_394656), then incubated in the dark at 4 ˚C for 30 min with 2.5 μL APC-conjugated TLR4/MD2 antibody (Thermo Fisher, #17-9924-82; RRID:AB_657858).

Techniques: Incubation, Infection, Mutagenesis