Fig 1: CD8 and LAG-3 immunohistochemistry in murine tumor tissues with FGL-1 silencing treated with anti-LAG-3 immunotherapy. CD8 immunohistochemistry in murine tumor tissues (a). LAG-3 immunohistochemistry in murine tumor tissues (b). The proportion of CD8+ cells in murine tumor tissues with ±FGL-1-sh and ±anti-LAG-3 (c). The proportion of LAG-3-positive cells in murine tumor tissues with ±FGL-1-sh and ±anti-LAG-3 (d). Arrows indicate positive cells. *p < 0.05, **p < 0.01; two-tailed Student’s t-test.
Fig 2: Lymphocyte activation gene-3 (LAG-3) knockdown promotes inflammatory activation by IFN-γ. BV2 cells were transfected with scramble control or LAG-3 siRNA. After incubation with the siRNAs, IFN-γ was added and the cells were harvested. (A) LAG-3 mRNA level was measured using quantitative real-time PCR to confirm the efficiency of knockdown by siRNA. (B) iNOS mRNA was measured in the siRNA-treated BV2 cells. (C) NO concentration was determined in culture supernatants of the siRNA-treated BV2 cells. Statistical analyses were performed using two-way ANOVA, followed by Tukey–Kramer post-hoc test; **p < 0.01, ****p < 0.0001. Error bars represent SEM.
Fig 3: STAT1 knockdown suppresses IFN-γ-induced LAG-3 expression in microglia. BV2 cells were transfected with scramble control or STAT1 siRNA. After incubation with the siRNAs, IFN-γ was added and the cells were harvested. (A) STAT1 mRNA level was measured using quantitative real-time PCR to confirm the efficiency of knockdown by siRNA. (B) LAG-3 mRNA was measured in the siRNA-treated BV2 cells. (C) LAG-3 expression in siRNA treated BV2 cells was analyzed by Western blotting. The image shown is representative of three independent experiments. Statistical analyses were performed using two-way ANOVA, followed by Tukey–Kramer post-hoc test; ****p < 0.0001. Error bars represent SEM.
Fig 4: IFN-γ induces membranous and soluble LAG-3 expression in microglia. (A) LAG-3 expression was analyzed by Western blotting in stimulated BV2 cells. (B) Representative images of LAG-3 expression in stimulated BV2 cells. Cells were immunofluorescent labeled with antibodies against LAG-3 or Iba1. Nuclei were stained by DAPI (scale bar, 20 μm). (C) Detection of LAG-3 at the plasma membrane by cell surface biotinylation assay. (D) Histogram showing the fluorescent intensity of cell surface LAG-3 in IFN-γ- or IL-13-stimulated BV2 cells by flow cytometry. The images shown are representative of three independent experiments.
Fig 5: Metalloproteases including ADAM10 and ADAM17 cleave membranous LAG-3 and produce soluble LAG-3 in microglia. BV2 cells were treated by indicated stimulants in the presence of following metalloproteinase inhibitors, after which cell lysate and culture supernatant were analyzed by Western blotting; (A) pan-metalloproteinase inhibitor GM6001, (B) ADAM10 inhibitor GI254023X, (C) ADAM17 inhibitor TAPI-1. Representative blots out of three experiments. (D) ADAM10 and ADAM17 mRNA expression was not changed in the presence of each inhibitor. Error bars represent SEM.
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