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: 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 transcription 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: The human lung cancer cell line A549 (ATCC, RRID: CVCL_4V07) and H292 (ATCC, RRID: CVCL_0455), mouse fibroblast cell line L929 (ATCC, RRID: CVCL_AR58), mouse lung adenocarcinoma cell line LLC (ATCC, RRID: CVCL_4358), mouse mononuclear macrophage leukemia cell line RAW264.7 (ATCC, RRID: CVCL_C6 × 3), and human embryonic kidney epithelial cell line 293T (ATCC, RRID: CVCL_0063) were procured from ATCC.
Techniques: Transfection, Co-Immunoprecipitation Assay, Phospho-proteomics, Western Blot, Software, Phagocytosis Assay, Flow Cytometry, Confocal Microscopy, Quantitative RT-PCR
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