Journal: European Journal of Immunology

Article Title: SYK Signalling in NLRP3 Inflammasome‐Mediated Response of Murine Microglia Activated by Immune Complexes Formed of Viral Proteins and Specific IgG

doi: 10.1002/eji.70199

Figure Lengend Snippet: SYK phosphorylation induced by VLPs and IC. Cells were treated with VLPs (20 µg/mL) and VLP‐specific mAbs (7.5 µg/mL) for 24 h, and 1 µM of SYK inhibitor R406 was added 1 h before treatment. (A) Fluorescent microscopy images of stained pSYK (green) and nuclei with Hoechst33342 (blue), pSYK was stained with primary Ab anti‐pSYK Y525/526 and secondary Ab‐AlexaFluor 488. The experiment was repeated four times, and representative composite images and MFI ± SD per cell of 30 frames are shown. The scale bar is 10 µm. (B) Western blot data show the expression of the indicated protein in primary microglia lysates, with representative images of four to six independent experiments. (C) Quantification of pSYK expression from Western blot data normalised to a loading control, N = 4–6. Data are represented using a box plot with dots showing the number of individual experiments. Significance was established using one‐way ANOVA followed by Tukey's test * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: SYK kinase inhibitor R406 (#inh‐r406), NLRP3 inhibitor MCC950 (#inh‐mcc), and Zymosan (#tlrl‐zyn) were from Invivogen.

Techniques: Phospho-proteomics, Microscopy, Staining, Western Blot, Expressing, Control

Journal: European Journal of Immunology

Article Title: SYK Signalling in NLRP3 Inflammasome‐Mediated Response of Murine Microglia Activated by Immune Complexes Formed of Viral Proteins and Specific IgG

doi: 10.1002/eji.70199

Figure Lengend Snippet: The impact of SYK inhibition on NLRP3 activation. Cells were treated with VLPs (20 µg/mL) and mAbs (7.5 µg/mL) for 24 h, and 1 µM of SYK inhibitor R406 or NLRP3 inhibitor MCC950 was added 1 h before treatment. (A) Western blot data show the expression of the indicated protein in microglia lysates, and the images are representative of five to six experiments. (B) Quantification of NLRP3 expression from Western blot data, normalised to a loading control, N = 5–6. (C) Quantification of ASC speck formation in primary microglia, N = 6. (D, E) IL‐1β secretion and (F) TNF‐α secretion were tested by ELISA in microglia supernatants, (D) N = 4, (E) N = 4–6, (F) N = 8–9. (G) Fluorescent microscopy images of the immunostained NLRP3 (cyan) and ASC specks (yellow), NLRP3 was stained with primary Ab anti‐NLRP and secondary Ab—AlexaFluor 488, ASC specks were stained with primary Ab anti‐ASC‐PE. Representative images are shown. The scale bars indicate 50 µm in large images and 20 µm in magnified images. In (B–F), data are represented using a box plot with dots showing the number of individual experiments. Significance was established using one‐way ANOVA followed by Tukey's test * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: SYK kinase inhibitor R406 (#inh‐r406), NLRP3 inhibitor MCC950 (#inh‐mcc), and Zymosan (#tlrl‐zyn) were from Invivogen.

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Control, Enzyme-linked Immunosorbent Assay, Microscopy, Staining

Journal: European Journal of Immunology

Article Title: SYK Signalling in NLRP3 Inflammasome‐Mediated Response of Murine Microglia Activated by Immune Complexes Formed of Viral Proteins and Specific IgG

doi: 10.1002/eji.70199

Figure Lengend Snippet: The impact of SYK inhibition on phagocytosis and antigen presentation. Cells were treated with pHrodo‐stained VLPs (10 µg/mL) and mAbs (7.5 µg/mL) for 3 h, 1 µM of SYK inhibitor R406 was added 1 h before treatment, and endocytosis inhibitor CytD was added for 30 min before treatment. pHrodo‐stained VLP and IC phagocytosis, CD86, and MHC II expression were measured by flow cytometry. (A) The representative gating strategy is shown. (B) Representative histograms of phagocytosis inhibited by CytD. (C) pHrodo mean fluorescent intensity (MFI) values normalised to isotype control, N = 3. (D) Phagocytosis index of pHrodo particles: data are presented as mean fluorescent intensity multiplied by % of cells with phagocytosed pHrodo particles, N = 7. (E, F) CD86 and MHC II MFI values normalised to isotype control of each experiment, (E) N = 8, (F) N = 11. In (C), data are represented as a bar graph as mean ± SD with dots showing the number of individual experiments. Statistical significance was established using Student's t ‐test. In (D–F), data are represented using box plots with dots showing the number of individual experiments. Statistical significance was established using one‐way ANOVA followed by Tukey's test * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: SYK kinase inhibitor R406 (#inh‐r406), NLRP3 inhibitor MCC950 (#inh‐mcc), and Zymosan (#tlrl‐zyn) were from Invivogen.

Techniques: Inhibition, Immunopeptidomics, Staining, Expressing, Flow Cytometry, Control

Journal: Cell Reports Medicine

Article Title: CAR-T triggers TAM reeducation and adaptive anti-tumor response via TREM2 deficiency or CD40 agonist

doi: 10.1016/j.xcrm.2025.102539

Figure Lengend Snippet: Trem2 -KO combined with IFN-γ promotes metabolic reprogramming in TAMs (A) Heatmap of KEGG pathways and differential gene set analysis of glycolysis, TCA, and fatty acid metabolism. Results were applied separately to Cxcl9 + and Spp1 + subtypes. (B) Heatmap of differential gene set analysis of glycolysis, TCA, and fatty acid metabolism. Results were applied separately to each group of CAR-T-treated WT or Trem2 -KO mice. (C) Schematic representation of in vitro tumor-educated macrophage model. BMDMs isolated from Trem2 -KO or WT mice were educated for 24 h in a transwell system with cancer cells, with or without IFN-γ (40 ng/mL) intervention. (D) Relative mRNA expression levels of Cxcl9 and Spp1 in BMDMs were quantified by RT-qPCR, and the ratio of Cxcl9 / Spp1 was calculated. (E) AMP and ATP concentrations in BMDMs were measured using dedicated assay kits, and the AMP/ATP ratio was calculated from these values. Relative mRNA expression levels of Ldha, Idh2, and Cpt1 in BMDMs were quantified by RT-qPCR. (F) Relative protein expression levels of indicated molecules were assessed by western blot (WB) and normalized to β-actin. (G, H, and O) BMDMs were pretreated for 2 h with the AMPK inhibitor compound C (20 μM; G), mTOR inhibitor rapamycin (1 μM; H), or SYK inhibitor R406 (1 μM; O), followed by 24 h co-culture with tumor cells in the presence or absence of IFN-γ (40 ng/mL). Protein expression was analyzed by WB and normalized to β-actin. (I and J) The real-time changes of OCR of BMDMs were stimulated with or without IFN-γ in the basal state and following the additions of oligomycin (Oligo), fluorocarbonyl cyanide phenylhydrazone (FCCP), etomoxir (Eto), and rotenone + antimycin A (Rot/AA). The average of basal OCR, maximal OCR, and Eto-sensitive OCR were revealed. (K and L) The real-time measurement of ECAR of BMDMs was stimulated with or without IFN-γ in the basal state and following the additions of glucose (Gluc), oligomycin (Oligo) and 2-deoxy-D-glucose (2-DG). The average of basal ECAR, maximal ECAR, and glycolytic reserve were revealed. (M and N) The relative protein expression levels of indicated molecular were assessed by WB and normalized to β-actin. (P) The iBMDMs were transiently transfected to overexpress SOCS1 for 48 h, followed by 24-h stimulation with or without IFN-γ (40 ng/mL). Indicated molecular protein expression levels were tested by WB and normalized to β-actin. Data were represented by mean ± SEM. ns, no significance, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: SYK inhibitor R406 , Selleck , Cat. #S1533.

Techniques: In Vitro, Isolation, Expressing, Quantitative RT-PCR, Western Blot, Co-Culture Assay, Transfection