pma  (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: LCMV‐infected Znfx1 mut mice show T cell expansion and more pronounced Th1 polarisation. (A) The white blood cell (WBC) count in whole blood. (B) The T cell count in whole blood. (C) Flow cytometry plot of CD62L and CD44 expression on CD4 + T cells in the spleen. EM: effector memory. (D) Flow cytometry plot of Ki‐67 expression on CD4 + T cells in the spleen. (E) Flow cytometry plot of T‐bet expression in CD4 + T cells in the spleen. (F) Flow cytometry plot of CXCR3 expression on CD4 + T cells in the spleen. (G) Flow cytometry plot of IFN‐γ expression in CD4 + T cells taken from the spleen after in vitro stimulation with 0.08 µM phorbol 12‐myristate 13‐acetate, 1.34 µM ionomycin, and brefeldin A for 5 h. (H) Flow cytometry plot of CXCR3 expression on CD8 + T cells in the spleen. (I) Flow cytometry plot of IFN‐γ expression in CD8 + T cells taken from the spleen after in vitro stimulation with 0.08 µM phorbol 12‐myristate 13‐acetate, 1.34 µM ionomycin, and brefeldin A for 5 h. (J) Flow cytometry plot of IFN‐γ expression in CD8 + T cells after in vitro stimulation with 10 µM gp33‐41 and brefeldin A for 5 h. All plots show cells from mice mock‐infected with NaCl or 9 days post infection (p.i.) with 200 PFU of LCMV strain WE. The data were analyzed in a one‐way analysis of variance with Šidák's correction for multiple comparisons. The graphs display the mean and the standard deviation. Each dot represents an individual mouse, and (except for the T cell count in blood) all plots show the data from at least two independent experiments. p ‐values are indicated above the plots.

Article Snippet: The cells were stimulated with 10 μM gp33‐41 (Med Chem Express), 10 μM gp61‐80 (Med Chem Express), or 0.08 μM phorbol 12‐myristate 13‐acetate (PMA), and 1.34 μM ionomycin (eBioscience Cell Stimulation Cocktail, Thermo Scientific).

Techniques: Infection, Cell Characterization, Flow Cytometry, Expressing, In Vitro, Standard Deviation

Journal: bioRxiv

Article Title: MDA5 multimerization on LINE RNA drives pathogenic extracellular immune complexes in autoimmunity

doi: 10.64898/2026.01.27.702129

Figure Lengend Snippet: a) (Upper) Volcano plot of differentially expressed genes in THP-1-derived macrophages (PMA + IFNγ/LPS) stimulated with MDA5ΔN filament-NP6 ICs over mock. All IC stimulations were performed without the use of intracellular delivery systems. Genes with log 2 -fold change |L2FC| >1 with p <0.05 (red) are shown; all other genes shown have p > 0.05 (gray) or p < 0.05 (blue). (Lower) Reactome pathways of significantly upregulated genes ( p adj < 0.05, L2FC > 0). b) Heatmap of Z -scored expression of interferon, interferon-stimulated genes, and other immune-related genes in THP-1-derived macrophages stimulated with combinations of MDA5ΔN (90nM monomer), 512 bp dsRNA (3 nM), mAb (NP6/NP8, 90 nM). Ctrl indicates isotype control Ab. IFIH1 and IFNB1 are highlighted. c) IFNB1 mRNA levels in THP-1-derived macrophages stimulated with combinations of MDA5ΔN, dsRNA, mAb (F01/F12, either full-length or Fab) as in (b). Data was normalized to mock treatment. P -values were calculated by two-tailed Student’s t test. d) Cellular and extracellular MDA5ΔN protein levels in THP-1-derived macrophages stimulated with MDA5ΔN monomer or filament ICs as in (b). 4 hours post-stimulation, supernatant and washed cell pellet were analyzed for MDA5ΔN, which was added extracellularly. e) IFNB1 mRNA levels in THP-1 macrophages pre-treated with PBS or Fc block for 1.5 hours, then stimulated with dsRNA-bound MDA5ΔN filaments (13.5 nM; 512 bp dsRNA, 0.45 nM) and NP6 Ab (13.5 nM). P -values were calculated by two-tailed Student’s t test. f) (Bottom) IFN-β ELISA using THP-1 macrophages electroporated with siRNAs prior to LPS/IFNγ priming and stimulated with dsRNA-bound MDA5ΔN filaments (90 nM; 512 bp dsRNA, 3 nM) and NP6 Ab (90 nM). (Top) Western blot showing knockdown of TRIF and MAVS proteins prior to IC stimulation. P -values were calculated by two-tailed Student’s t test. g) IFNB1 mRNA levels in THP-1 macrophages stimulated with ICs composed of NP6/NP8 Abs (90 nM), MDA5ΔN (90 nM), and 512 bp dsRNA (3 nM, left; 0.9 nM, middle; 0.3 nM, right). Data was normalized to mock treatment. P -values were calculated by two-way ANOVA to compare NP6 and NP8 IC values across 4 mAb concentrations. Data are presented as mean ± SD of 3 biological replicates. RNA-seq results contain 2 biological repeats. All other data are representative of at least 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. n.s , not significant.

Article Snippet: Following adherence, neutrophils were stimulated with either vehicle control, 4 μM ionomycin (Invivogen), or 100nM PMA (Invivogen) for 4 hours at 37°C and 5% CO2.

Techniques: Derivative Assay, Expressing, Control, Two Tailed Test, Blocking Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Knockdown, RNA Sequencing