Journal: Frontiers in immunology

Article Title: Aspartate Metabolism Facilitates IL-1β Production in Inflammatory Macrophages.

doi: 10.3389/fimmu.2021.753092

Figure Lengend Snippet: FIGURE 4 | Asparagine promotes the production of interleukin 1b (IL-1b) and the activation of signal pathways in macrophages. (A) IL-1b and tumor necrosis factor alpha (TNF-a) secretion in M1 macrophages and asparagine-treated M1 macrophages (n = 4–5). (B) mRNA expressions of IL-1b and TNF-a in M1 macrophages and asparagine-treated M1 macrophages (n = 6). (C–E) Protein abundance of IL-1b, IkB kinase (IKK), p-IKK, IkB, p-IkB, p65, p-65, hypoxia-inducible factor-1a (HIF- 1a), Nod-like receptor protein 3 (NLRP3), apoptosis-associated speck like protein containing a caspase recruitment domin (ASC), and caspase-1 in macrophages (n = 4). (F) IL-1b secretion in M1 macrophages with asparagine combined with IKK16 or VX-765 (n = 3). Data were analyzed with unpaired t-test (A–E) or with one- way ANOVA (F). Error bars represent the mean ± SEM. *p ≤0.05, **p ≤0.01.

Article Snippet: PX-478, MCC-950, VX-765, and IKK16 were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Activation Assay, Quantitative Proteomics

Journal: Scientific Reports

Article Title: A phenotypic high-content, high-throughput screen identifies inhibitors of NLRP3 inflammasome activation

doi: 10.1038/s41598-021-94850-w

Figure Lengend Snippet: NLRP3 inflammasome inhibitor hits validation in the speck assay. ( a ) Schematic of the priming protocol. ( b ) Schematic of the activation protocol. ASC-mCherry iBMDM cells were treated with indicated concentrations of ( c ) PU H71, ( d ) MPC-3100, ( e ) momelotinib, ( f ) CEP-33779, ( g ) ACHP or ( h ) MLN120B with LPS (1 µg/ml) for 2 h followed by nigericin treatment (10 µM, 2 h) after which PFA was added (priming—blue trace) and specks counted. In a parallel experiment the compounds were added after LPS treatment just prior to nigericin (activation—red trace). The images are representative of the nuclei and speck formation for each compound at 10 µM in both priming and activation protocols. Data are presented as mean ± SEM, n = 3 independent experiments. Data in ( c – h ) was analyzed using GraphPad Prism version 7 software ( https://www.graphpad.com/scientific-software/prism/ ).

Article Snippet: MCC950 (CAS number: 256373-96-3) was purchased from Tocris; PU H71 (Cat. number: 1856) and momelotinib (CAS number: 1056634-68-4) were purchased from Axon Medchem; MPC-3100 (Cat. number: A4063) was purchased from ApexBio; MLN120B (CAS number: 783348-36-7), CEP-33779 (CAS number: 958025-66-6) and ACHP (CAS number: 406209-26-5) were purchased from MedChemExpress.

Techniques: Biomarker Discovery, Activation Assay, Software

Journal: Frontiers in Immunology

Article Title: Nuclear Factor κB is Required for Tumor Growth Inhibition Mediated by Enavatuzumab (PDL192), a Humanized Monoclonal Antibody to TweakR

doi: 10.3389/fimmu.2013.00505

Figure Lengend Snippet: Inhibition of NFκB activation prevents the growth inhibitory activity of enavatuzumab . (A) BT549 cells were transfected with two different siRNAs against p65 or upstream kinases IKK1 (IκBKβ), IKK2 (CHUK). Transfection controls were non-targeting control (negative) or KSP (positive control for transfection efficiency). After 48 h, cells were treated with enavatuzumab or IgG1 control at 10 μg/mL for an additional 5 days in the presence of anti-human crosslinking antibody (3.5 μg/mL), and the cell viability was determined. siRNA significantly reduced growth inhibition by enavatuzumab, compared to mock or control siRNA transfected cells (horizontal line) (* p -value < 0.05). (B) Enavatuzumab sensitive lines were treated with IKK inhibitor IKK16 at 160 nM (black bars), enavatuzumab/crosslinker (gray bars), or IKK16 plus enavatuzumab/crosslinker (striped bars) and % survival was measured after 5 days. IKK16 significantly blocked growth inhibition by enavatuzumab compared to enavatuzumab alone (* p -value < 0.05). (C) Targeting individual NFκB subunits (p50, p65, RelB, p52) by siRNA reduced enavatuzumab activity in sensitive cell lines (MDA-MB-468 and BT549). Cells were transfected with pooled targeting siRNA or control siRNA for 48 h, prior to treatment with enavatuzumab or IgG1 control for 5 days in the presence of a crosslinking antibody. Percent survival was calculated from the relative viability of cells treated with enavatuzumab versus control-treated cells (* p -value < 0.05). (D) The effect of NFκB induced by enavatuzumab on cell division was investigated by pre-labeling HT3 cells with the Cell Trace™ reagent. Cells treated with enavatuzumab/crosslinker (green line) are compared to the IgG1 control-treated cells (red line). Cells transfected with two different siRNAs to p65, p50, p52, or RelB and treated with enavatuzumab/crosslinker (blue and brown lines) are also displayed.

Article Snippet: The IκB kinase (IKK) selective inhibitor IKK16 (Tocris Bioscience) was used at 160 nM ( ).

Techniques: Inhibition, Activation Assay, Activity Assay, Transfection, Control, Positive Control, Labeling