poly i c  (InvivoGen)

Journal: European Journal of Immunology

Article Title: A Novel Murine Model of Hemophagocytic Lymphohistiocytosis‐Like Inflammation in ZNFX1 Deficiency

doi: 10.1002/eji.70141

Figure Lengend Snippet: BMDMs from Znfx1 mut mice produce more pro‐inflammatory cytokines in an unstimulated state. (A) Schematic representation of the culture and stimulation of bone marrow‐derived macrophages (BMDMs). (B) TNF‐⍺ expression of BMDMs from WT and Znfx1 mut mice in an unstimulated control setting or after stimulation with 2 µg/mL Poly(I:C) HMW for 8 h, with brefeldin A for 6 h. (C) IL‐6 expression of BMDMs from WT and Znfx1 mut mice in an unstimulated control setting or after stimulation with 2 µg/mL Poly(I:C) HMW for 8 h, with brefeldin A for 6 h. (D) IFN‐γ expression of BMDM from WT and Znfx1 mut mice in an unstimulated control setting or after stimulation with 2 µg/mL Poly(I:C) HMW for 8 h, with brefeldin A for 6 h. Representative images are shown in panel (i). The measured log10 single‐cell intensity values are combined into violin plots for each mouse, and the number of measured cells per mouse is stated beneath the respective plot in panel (ii). The values for all the mice in the same group were grouped into violin plots in panel (iii). For the statistical analysis, a separate Gaussian mixture model (GMM) was fitted with the cell intensity values for each mouse. A weighted group mean and the covariance were calculated from the number of observations per mouse. Panel (iv) shows the resulting histogram for unstimulated WT cells with the combination of the weighted GMMs for the entire group (black line) and the three components of the fit (shown in dark blue, light blue, and yellow). The means were compared in a two‐tailed t ‐test; p ‐values are indicated. The data represent the results of one experiment, n = 3.

Article Snippet: To stimulate cytokine production, medium with 2 μg/mL Poly(I:C) HMW (InvivoGen) was added for a total of 8 h. BD GolgiPlug protein transport inhibitor (BD) was added to all wells for a total of 6 h. Cells were fixed with prewarmed paraformaldehyde (Electron Microscopy Sciences, end concentration: 4%) for 30 min at room temperature.

Techniques: Derivative Assay, Expressing, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Two orthogonal MAP3K-driven pathways of NLRP1 inflammasome activation revealed by poisonous beetles

doi: 10.64898/2026.01.23.701189

Figure Lengend Snippet: Poly I:C and CHIKV activates the NLRP1 inflammasome via combined actions of TAK1 and ZAKα. A. IL-18 ELISA or B. Immunoblots of cleaved IL-1β, Pro-IL-1β, IL-18 (full length and cleaved), and GAPDH from N/TERTs pre-treated with 1 μM 5-z7-oxozeanol (pan-kinase inhibitor), 1 μM 6p (ZAK inhibitor), or DMSO (vehicle control) before stimulation with 200ng/mL Poly I:C. Cells and cell culture media were harvested after 8 hours of treatment. C. Percentage of ASC-GFP speck forming A549 NLRP1 ASC-GFP reporter cells pre-treated with 1 μM 5-z7-oxoxeanol (pan-kinase inhibitor), 1 μM 6p (ZAK inhibitor), or DMSO (vehicle control) before stimulation with 200 ng/mL Poly I:C. Cells were fixed and stained with DAPI after 4 hours of treatment. D. Rhodamine labelled SNAP-tagged NLRP1-DR, immunoblot of ZAK and p38 following PhosTag-SDS-Agarose-PAGE, and immunoblot of p-JNK, t-JNK, and GAPDH following SDS-PAGE from N/TERTs pre-treated with 5 μM emricasan (pan-caspase inhibitor), 1 μM 5-z7-oxoxeanol (pan-kinase inhibitor), 1 μM 6p (ZAK inhibitor), or DMSO (vehicle control) before stimulation with 1 μM anisomycin, DMSO (vehicle control), 200 ng/mL Poly I:C, and LFK2 (transfection control). Cell lysates were harvested after 6 hours of treatment. E. Rhodamine labelled SNAP-tagged NLRP1-DR, immunoblot of ZAK and p38 following PhosTag-SDS-Agarose-PAGE, and immunoblot of p-JNK, t-JNK, and GAPDH following SDS-PAGE from TAK1 KO, ZAKα KO, T+Z dKO, and RNP control N/TERTs expressing NLRP1-86-275-SNAP stimulated with 200 ng/mL Poly I:C, LFK2 (transfection control), and 1 μM anisomycin. Cells were harvested after 6 hours of treatment. F. IL-18 ELISA from TAK1 KO, ZAKα KO, T+Z dKO, and RNP control N/TERTs following treatment with 200 ng/mL Poly I:C, LFK2 (transfection control), and 1 μM anisomycin. Cell culture media was harvested after 8 hours of treatment. G. Immunoblot of GSDMD (full length and cleaved), GSDME (full length and cleaved), GAPDH, cleaved IL-1β, Pro-IL-1β, and IL-18 (full length and cleaved) of NLRP1 KO N/TERTs overexpressing GFP_NLRP1_86-C_FLAG (WT), TZ2 mutant, or GFP control. Cells, floaters and cell culture media were harvested after 8 hours of treatment. H. IL-18 ELISA of ASC KO N/TERTs overexpressing ASC-GFP (N/TERT ASC-GFP reporter cells) pre-treated with 1 μM 5-z7-oxozeanol (pan-kinase inhibitor), 1 μM 6p (ZAK inhibitor), or DMSO (vehicle control) before stimulation with CHIKV (MOI 10). Cell culture media was harvested after 16 hours of treatment. I. Percentage of ASC-GFP speck forming TAK1 KO, ZAKα KO, T+Z dKO, and RNP control N/TERT ASC-GFP reporter cells following treatment with CHIKV (MOI 10), mock (vehicle control for CHIKV), 200 ng/mL Poly I:C, and LF2K (transfection control). Cells were fixed and stained with DAPI after 14 hours of treatment. J. Immunoblot of MX1 (IFN-stimulated gene), Pro-IL-1β, cleaved IL-1β, IL-18 (full length and cleaved), and GAPDH of TAK1 KO, ZAKα KO, T+Z dKO, and RNP control N/TERTs following treatment CHIKV (MOI 10) and mock (vehicle control for CHIKV). Cells and cell culture media were harvested after 14 hours of treatment. K. Schematic of known dsRNA sensors and downstream signalling cascades. 3p-hpRNA is a RIG-I agonist while 2-5A activates RNaseL. RIG-I and RNaseL are upstream of TAK1 and ZAKα respectively. L. Percentage of ASC-GFP speck forming A549 NLRP1 ASC-GFP reporter cells following transfection of 3p-hpRNA and 2-5A individually or in combination. Cells were fixed and stained with DAPI after 3 hours of treatment. Error bars represent SEM from three technical replicates, where one replicate refers to an independent sample. Significance values were calculated based on one-way ANOVA followed by Dunnett’s test (A, C, and H) or two-way ANOVA followed by Sidak’s test for multiple pairwise comparisons (F, I, and L). ns, nonsignificant; *P < 0.05; ****P < 0.0001.

Article Snippet: Cells were transfected with HMW Poly(I:C) (InvivoGen), 3php (InvivoGen), and compound 2-5A using Lipofectamine 2000 (Thermo Fisher Scientific, 11668019) diluted in Opti-MEM.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Control, Cell Culture, Staining, SDS Page, Transfection, Expressing, Mutagenesis