Journal: Nature Immunology

Article Title: Human T H 17 cells engage gasdermin E pores to release IL-1α on NLRP3 inflammasome activation

doi: 10.1038/s41590-022-01386-w

Figure Lengend Snippet: a , Lane views of electropherograms obtained with the Jess Simple Western System (ProteinSimple). Monocytes were MACS-isolated with CD14 beads and stimulated in the presence or absence of LPS (24 h) and nigericin (Nig.) for 30 min before cell lysis. Th17 cells were FACS-sorted ex vivo from the blood of healthy donors, stimulated for 48 h and transfected with RNP containing nontargeted control (NTC) or crASC. Cell lysates were collected after 7 days. The experiment was repeated independently 3 times with similar results. b , Western blot analysis. Cell culture lysates derived from Th17 cells and monocytes. Th17 cells were sorted as in (a) and stimulated with anti-CD3 and anti-CD28 mAbs (48 h plate-bound) for 5 days. Monocytes were isolated and stimulated as in (a). The experiment was repeated independently 2 times with similar results. c , NLRP3-speck formation assessed by ImageStream in Th17 cells stimulated for 5 days with anti-CD3 and anti-CD28 mAbs. Each circle indicates an individual healthy blood donor and experiment. Two-sided paired t-test. Data are presented as mean ± SEM. n = 4 biologically independent samples examined over 2 independent experiments.

Article Snippet: In some experiments, CD14 + magnetic activated cell sorting (MACS)-sorted monocytes were differentiated into macrophages for 7 d in the presence of granulocyte–macrophage colony-stimulating factor (R&D Systems).

Techniques: Simple Western, Isolation, Lysis, Ex Vivo, Transfection, Control, Western Blot, Cell Culture, Derivative Assay

Journal: Nature Immunology

Article Title: Human T H 17 cells engage gasdermin E pores to release IL-1α on NLRP3 inflammasome activation

doi: 10.1038/s41590-022-01386-w

Figure Lengend Snippet: a , Differential gene expression determined by transcriptome analysis of T H 17 cells treated as in Fig. ( n = 3 individual healthy blood donors). b , RT–qPCR analysis of anti-CD3 and anti-CD28 monoclonal antibody-stimulated, naive T cells in polarizing cytokine conditions ( n = 4, one-way ANOVA with Dunnett’s multiple-comparison test). c , Immunoblot analysis of cell lysates from T H 17 cells stimulated with anti-CD3 and anti-CD28 monoclonal antibodies for different durations. The data represent three experiments. d , ELISA of cell culture supernatants from T H 17 cells with and without deletion of GSDME (left) or GSDMD (right) by CRISPR–Cas9 technology. Individual experiments were normalized to the first time point of analysis on day 2 ( n = 3 individual biological samples, two-way ANOVA with Bonferroni’s multiple-comparison test). e , Immunoblot analysis of cell lysates from T H 17 cells stimulated with anti-CD3 and anti-CD28 monoclonal antibodies for the indicated time points and of CD14 + monocytes stimulated for 24 h with LPS and 30 min with nigericin. Casp, Caspase. f , Lane view of electropherograms obtained with a Jess Simple Western System for cell lysates of T H 17 cells stimulated for 5 d as in e in the presence or absence of the indicated inhibitors. It is a representative experiment. g , Cumulative data of f (one-sample Student’s t -test). AUC, area under the curve. h , Luminex assay of the supernatants of T H 17 cells stimulated with plate-bound anti-CD3 (1 μg ml −1 , TR66) and phorbol-12,13-dibutyrate for 8 h on day 4 of culture ( n = 3 individual biological samples, two-tailed, paired Student’s t -test). i , ELISA of supernatants of T H 17 cells stimulated as in f . Each circle indicates an independent blood donor in h and i ( n = 4 individual biological samples; two-tailed, paired Student’s t -test).

Article Snippet: In some experiments, CD14 + magnetic activated cell sorting (MACS)-sorted monocytes were differentiated into macrophages for 7 d in the presence of granulocyte–macrophage colony-stimulating factor (R&D Systems).

Techniques: Gene Expression, Quantitative RT-PCR, Comparison, Western Blot, Bioprocessing, Enzyme-linked Immunosorbent Assay, Cell Culture, CRISPR, Simple Western, Luminex, Two Tailed Test