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. 2019 Sep 18;10:2243. doi: 10.3389/fimmu.2019.02243

Figure 5.

Figure 5

RAGE and TLR4 receptors are responsible for S100A9-mediated MDSCs chemotaxis and activation. (A,B) Real-time quantitation PCR analysis of RAGE (A) and TLR4 (B) mRNA expression in LoVo-induced MDSCs treated with GST-S100A9 (20 μg/ml) and GST (20 μg/ml) for 3, 12, and 24 h. (C) Western blot analysis of RAGE and TLR4 expression in MDSCs treated with GST-S100A9 (20 μg/ml) and GST (20 μg/ml) for 24 h. (D) Chemotaxis assay for LoVo-induced MDSCs pretreated with and without inhibitors FPS-ZM1 (100 nM) and TAK-242 (100 nM) for 30 min followed by stimulation with GST-S100A9 (20 μg/ml) and GST (20 μg/ml) proteins for 24 h. (E–G) Real-time quantitative PCR analysis for mRNA expression of immunosuppressive molecules, including Arg1 (E), iNOS (F), and IL10 (G) in LoVo-induced MDSCs pretreated with and without inhibitors FPS-ZM1 (100 nM) and TAK-242 (100 nM) for 30 min followed by stimulation with GST-S100A9 (20 μg/ml) and GST (20 μg/ml) proteins for 24 h. (H) Fluorescence intensity analysis for ROS production in LoVo-induced MDSCs pretreated with and without inhibitors FPS-ZM1 (100 nM) and TAK-242 (100 nM) for 30 min followed by stimulation with GST-S100A9 (20 μg/ml) and GST (20 μg/ml) proteins for 24 h. (I) Suppression of LoVo-induced MDSCs pretreated with and without inhibitors FPS-ZM1 (100 nM) and TAK-242 (100 nM) for 30 min followed by stimulation with GST-S100A9 (20 μg/ml) and GST (20 μg/ml) proteins on T cells in vitro. (J) A statistical graph of the suppressive effect of LoVo-induced MDSCs pretreated with and without inhibitors FPS-ZM1 (100 nM) and TAK-242 (100 nM) for 30 min followed by stimulation with GST-S100A9 (20 μg/ml) and GST (20 μg/ml) proteins on CD8+ T cells. Data represent three independent experiments and are represented as Mean±SD. *p < 0.05, **p < 0.01, ***p < 0.001.