Journal: Journal of experimental & clinical cancer research : CR
Article Title: Galectin-3 induces pathogenic immunosuppressive macrophages through interaction with TREM2 in lung cancer.
doi: 10.1186/s13046-024-03124-6
Figure Lengend Snippet: Fig. 4 Galectin-3 inhibits TREM2/DAP12 receptor complex to suppress Src/Syk signaling pathway and altered macrophage to an M2-like phenotype. (A, B) 293T cells were transfected with plasmids as shown, and anti-HA (B) or anti-Flag (B) antibodies were employed for exogenous CO-IP experiments. (C-E) 293T cells were transfected with plasmids as shown and anti-HA (C), anti-Flag (D), or anti-Myc (E) antibodies were employed for exogenous CO-IP experiments, respectively. (F, G) After 24 h of LLC CM treatment with the addition of GB1107 (5 µM), and 2 h of stimulation with LPS (1 ng/mL), the phosphorylation levels of Src and Syk in indicated time point were analyzed by western blot (F). The gray values of p-Syk and p-Src protein bands were analyzed by Image J software, and the relative gray values were standardized to the gray values of Syk and Src (G). (H, I) After 24 h of LLC CM treatment with the addition of GB1107 (5 µM), and 2 h of stimulation with LPS (1 ng/mL), the phosphorylation levels of Src and Syk in WT or TREM2 KO BMDMs were analyzed by western blot (H). The gray values of p-Syk and p-Src protein bands were analyzed by Image J image analyses software, and the relative gray values were standardized to the gray values of Syk and Src (I). (J, K) After 24 h of LLC CM treatment with the addition of rm galectin-3 (200 ng/ml), and 2 h of stimulation with LPS (1 ng/mL), the phosphorylation levels of Src and Syk in WT or TREM2 KO BMDMs were analyzed by western blot (J). The gray values of p-Syk and p-Src protein bands were analyzed by Image J image analyses software, and the relative gray values were standardized to the gray values of Syk and Src (K). (L) Phagocytosis assay between WT or TREM2 KO BMDMs which were pretreated with LLC CM supplemented with GB1107 (5 µM) for 24 h and LLC were detected by flow cytometry. (M) Following a 24 h-treatment with LLC CM supplemented with GB1107 (5 µM), the F-actin polarization of RAW264.7 cells blocked with anti-TREM2 antibody was observed by confocal microscopy. Scale bars, 5 μm. Quantitative statistics of F-actin polarization were analyzed by Image J image analyses software. (N-P) After 24 h of LLC CM which was supplemented with GB1107 (5 µM) treatment, the transcrip tion levels of Ccr2(N), M2-like macrophage markers (CD206, Arg1) (O) and M1-like macrophage markers (Nos2, TNFα) (P) in WT or TREM2 KO BMDMs were detected by RT-qPCR assay. Data represent mean ± SD from three experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, no significance
Article Snippet: Agonists and inhibitors Tyrosine kinase inhibitor (Genistein) (absin, Shanghai, China), Syk inhibitor (R406) (Selleck Chemicals, Houston, USA), Src inhibitor (PP2) (Solarbio, Beijing, China), TREM2 Fc (R&D Systems, Minnesota, USA), GB1107 (Selective Galectin-3 inhibitor) (TargetMol, Boston, Massachusetts), Recombinant mouse Galectin-3 protein (rm Galectin-3) (R&D Systems, Minnesota, USA).
Techniques: Transfection, Co-Immunoprecipitation Assay, Phospho-proteomics, Western Blot, Software, Phagocytosis Assay, Flow Cytometry, Confocal Microscopy, Quantitative RT-PCR
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