Journal: Life Science Alliance

Article Title: Caspase-1 interdomain linker cleavage is required for pyroptosis

doi: 10.26508/lsa.202000664

Figure Lengend Snippet: (A, B) HEK 293T cells stably expressing the indicated pro-caspase-1 constructs were transiently transfected with plasmids encoding CARD8-Flag (0.05 μg) or NLRP1-Flag (0.1 μg) and ASC (0.01 μg) without GSDMD (A) or with GSDMD (0.01 μg) (B). After 24 h, the cells were treated with VbP (10 μM, 6 h) before lysates were analyzed by immunoblotting. (C) HEK 293T cells stably expressing the indicated pro-caspase-1 were transiently transfected with residues 1–328 of NLRC4 for 24 h before lysates were evaluated by immunoblotting. (D) Schematic of the DmrB-caspase-1 constructs. Predicted cleavage sites, sizes of potential cleavage products, and the catalytic cysteine are indicated. (E) HEK 293T cells stably expressing GSDMD were transiently transfected with the indicated DmrB-caspase-1 constructs for 24 h before addition of AP-20187 (500 nM, 1 h). GSDMD and CASP1 cleavage were evaluated by immunoblotting. All data, including immunoblots, are representative of three or more independent experiments. CL, cleaved; FL, full-length.

Article Snippet: A plasmid encoding NLRC4 was purchased from Origene (RC206757) and a construct encoding residues 1–328 was cloned into the Gateway system.

Techniques: Stable Transfection, Expressing, Construct, Transfection, Western Blot

Journal: Cell Death & Disease

Article Title: Sensing soluble uric acid by Naip1-Nlrp3 platform

doi: 10.1038/s41419-021-03445-w

Figure Lengend Snippet: A Uric acid levels measured in the serum of humans, mice and rhesus monkeys. IL-1β Elisa of B monocyte-derived macrophages collected from healthy people, C murine bone marrow-derived macrophages and D monocyte-derived macrophages collected from rhesus monkeys, all stimulated under different conditions. In B – D , LPS was added for 1 h at 100 ng/mL and the media were posteriorly changed. MSU (100 μg/mL) and sUA (200 μΜ) were added for 6 h. Each coloured dot represents a different individual. E IL-1β Elisa of BMDM derived from Naip1 −/− , Naip2 −/− , Naip5 −/− , ΔNaip −/− and Nlrc4 −/− mice under different stimulus. F IL-1β Elisa of human THP1 cells virally transduced with plasmids carrying Naip1, Naip5, Naip6, Nlrc4 and empty vector using lentivirus constructs and posteriorly stimulated under different conditions. In E , F , LPS was added for 1 h at 100 ng/mL and the media were posteriorly changed. sUA (200 μΜ) was added for 6 h and nigericin (10 μΜ) was added for 90 min. In A , n = 5 for each analysed species. Triangle refers to human’s sample, Circle refers to murine’s sample and Square refers to rhesus’ sample. In B , C , we collected cells from eight different individuals; in D , we collected cells from seven different individuals. In E , F , data are plotted as the median of a triplicate of three to four independent experiments. * p < 0.05 and *** p < 0.001; n.s. not significant.

Article Snippet: Plasmids for GFP-tagged mNaip1 (#60200), Naip5 (#60205), Naip6 (#60202), Nlrc4 (#60199) and empty vector (#60206) were purchased from Addgene (Watertown, MA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Transduction, Plasmid Preparation, Construct

Journal: Journal of Autoimmunity

Article Title: Prophylactic and therapeutic treatment with a synthetic analogue of a parasitic worm product prevents experimental arthritis and inhibits IL-1β production via NRF2-mediated counter-regulation of the inflammasome

doi: 10.1016/j.jaut.2015.04.005

Figure Lengend Snippet: SMA-12b downregulates genes associated with production of bioactive IL-1β in bmMs . The effect of exposure of bmMs to SMAs-11a and -12b (both at 5 μg/ml) over 4 h on the steady state- and LPS-induced mRNA levels of IL-1β (A & D); NLRP3 (B & E) and NLRC4 (C & F) as assessed by qRT-PCR where the levels of the gene of interest were normalized to the level of GAPDH and expressed as a fold change with respect to the medium control. Data are presented as the means ± SEM of the mean of replicate values pooled from 3 individual experiments. *p < 0.05; **p < 0.01 and ***p < 0.001. Black* represent significance between 12b (or 11a) and control whereas grey* represents significant differences between 12b- and 11a-treated cells.

Article Snippet: TaqMan ® RT-PCR was performed using the following TaqMan ® Gene Expression Assays: IL-1β (Mm01336189_m1), chemokine receptor 5 (CCR5: Mm01216171_m1), chemokine receptor 2 (CCR2: Mm00438270_m1), chemokine ligand 10 (CXCL10; Mm00445235_m1), complement component 5a receptor 1 (C5AR1; Mm00500292_s1), CD274 (PD-L1) (Mm00452054_m1), CD200 receptor 1 (CD200R1: Mm02605260_s1), NLRP3 (Mm00840904_ml), NLRC4 (Mm01233149_ml), glutamate-cysteine ligase, modifier subunit (GCLM; Mm00514996_ml), glutamate-cysteine ligase, catalytic subunit (GCLC: Mm00802655_ml) and haem oxygenase 1 (HMOX1; Mm00516005_ml), all from Applied Biosystems.

Techniques: Quantitative RT-PCR, Control

Journal: Journal of Autoimmunity

Article Title: Prophylactic and therapeutic treatment with a synthetic analogue of a parasitic worm product prevents experimental arthritis and inhibits IL-1β production via NRF2-mediated counter-regulation of the inflammasome

doi: 10.1016/j.jaut.2015.04.005

Figure Lengend Snippet: SMA-12b-mediated changes in gene expression are abrogated in NRF2-deficient bmMs . The effect of exposure (4 h) of bmMs from wild type and NRF2-deficient (NRF2 KO) C57BL/6 mice to SMA-12b on the mRNA levels of IL-1β (A; n = 5); NLRP3 (B; n = 5); NLRC4 (C; n = 6) and GCLC (D; n = 6) as assessed by qRT-PCR. The levels of the gene of interest were normalized to GAPDH and expressed as a fold change with respect to the relevant wild type medium control. Data are presented as the means ± SEM, where n represents matched replicate cultures of individual wild type and KO mice. *p < 0.05; **p < 0.01 and ***p < 0.001 where significance is for WT SMA treatments relative to the wild type “none” condition as indicated by black* and grey* indicates significance between “none” WT and “none” KO samples.

Article Snippet: TaqMan ® RT-PCR was performed using the following TaqMan ® Gene Expression Assays: IL-1β (Mm01336189_m1), chemokine receptor 5 (CCR5: Mm01216171_m1), chemokine receptor 2 (CCR2: Mm00438270_m1), chemokine ligand 10 (CXCL10; Mm00445235_m1), complement component 5a receptor 1 (C5AR1; Mm00500292_s1), CD274 (PD-L1) (Mm00452054_m1), CD200 receptor 1 (CD200R1: Mm02605260_s1), NLRP3 (Mm00840904_ml), NLRC4 (Mm01233149_ml), glutamate-cysteine ligase, modifier subunit (GCLM; Mm00514996_ml), glutamate-cysteine ligase, catalytic subunit (GCLC: Mm00802655_ml) and haem oxygenase 1 (HMOX1; Mm00516005_ml), all from Applied Biosystems.

Techniques: Gene Expression, Quantitative RT-PCR, Control