Fig 1: Hypoxic glioma cell-derived EVs promote M2 macrophage polarization. (A) Images of EVs observed under a TEM (Scale bar = 100 nm). (B) Diameter of EVs detected by dynamic light scattering. (C) The expression of CD9, CD63, TSG101, and calnexin on the surface of EVs determined by Immunoblotting. (D) Detection of the uptake of EVs from normoxic and hypoxic glioma cells by macrophages U937 through immunofluorescence (400×). (E) The mRNA expression of iNOS, TNF-a, Arg-1, and IL-10 assessed by RT-qPCR after EV treatment. (F) Immunoblotting of protein expression of iNOS and Arg-1 in macrophages U937 after EV treatment. (G) The expression of TNF-a and IL-10 in supernatant of macrophages U937 evaluated by ELISA assay. (H) The proportion of CD11b+CD163+ cells in U937 cells examined by flow cytometry. *p < 0.05 versus macrophages treated with PBS. # p < 0.05 versus macrophages treated with normal oxygen-induced glioma cell-derived EVs. The experiment was repeated three times
Fig 2: NEDD4L downregulates PIK3CA to inhibit the PI3K/AKT pathway, thus restraining M2 polarization of macrophages and glioma cell proliferation, migration, and invasion. (A) The protein expression of NEDD4L, PIK3CA, and AKT and phosphorylated AKT in U937 cells after overexpression NEDD4L and PIK3CA examined by Immunoblotting. (B) The mRNA expression of iNOS, TNF-a, Arg-1, and IL-10 in U937 cells after overexpression NEDD4L and PIK3CA measured by RT-qPCR. (C) Immunoblotting of protein expression of iNOS and Arg-1 in U937 cells after overexpression NEDD4L and PIK3CA. (D) The expression of TNF-a and IL-10 in supernatant of U937 cells after overexpression NEDD4L and PIK3CA assessed by ELISA. (E) The proportion of CD11b+CD163+ cells in U937 cells after overexpression NEDD4L and PIK3CA evaluated by flow cytometry. (F) The proliferation in U87 and A172 cells co-cultivated with U937 cells tested by EdU. (G) Cell migration in U87 and A172 cells co-cultivated with U937 cells examined by Transwell. (H) Cell invasion in U87 and A172 cells co-cultivated with U937 cells examined by Transwell. *p < 0.05 versus U937 cells transfected with oe-NC or U87 or A172 co-cultivated with U937 cells treated without anything. # p < 0.05 versus U937 cells transfected with oe-PIK3CA or U87 or A172 co-cultivated with U937 cells transfected with oe-PIK3CA. The experiment was repeated three times
Fig 3: The effects of avicularin on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in bradykinin-treated MG-63 human osteoblastic osteosarcoma cells. MG-63 cells were treated with increasing concentrations of avicularin for 48 h, and 1 µM of bradykinin was used to treat the cells for 24 h. (A) Western blot assay was performed to measure the protein expression of iNOS and COX-2. (B) The ratio of iNOS/GAPDH is presented. (C) The ratio of COX-2 GAPDH is presented. Quantitative real-time polymerase chain reaction (qRT-PCR) detected the expression level of iNOS (D) and COX-2 (E) at the mRNA level. Data are presented as the mean±standard deviation (SD) from three independent experiments. ** p<0.01 vs. the control group; #, ## p<0.05, p<0.01 vs. the bradykinin group.
Fig 4: miR-10b-5p stimulates M2 polarization of macrophages to increase proliferation, migration, and invasion of glioma cells via downregulation of NEDD4L. (A) The expression of miR-10b-5p and NEDD4L in U937 cells after overexpression of miR-10b-5p and NEDD4L measured by RT-qPCR. (B) The mRNA expression of iNOS, TNF-a, Arg-1, and IL-10 in U937 cells after overexpression of miR-10b-5p and NEDD4L determined by RT-qPCR. (C) The protein expression of iNOS and Arg-1 in U937 cells after overexpression of miR-10b-5p and NEDD4L examined by Immunoblotting. (D) The expression of TNF-a and IL-10 assessed in supernatant of U937 cells after overexpression of miR-10b-5p and NEDD4L examined by ELISA. (E) The proportion of CD11b+CD163+ cells in U937 cells after overexpression of miR-10b-5p and NEDD4L evaluated by flow cytometry. (F) The proliferation in glioma U87 and A172 cells co-cultured with transfected U937 cells assessed by EdU. (G) Cell migration in U87, and A172 cells co-cultured with U937 cells tested by Transwell. H, Cell invasion in U87 and A172 cells co-cultured with U937 cells tested by Transwell. *p < 0.05 versus U937 cells transfected with Vector and mimic-NC or U87 or A172 co-cultured with U937 cells treated without anything. # p < 0.05 versus U937 cells transfected with NEDD4L and mimic-NC or U87 or A172 co-cultured with U937 cells transfected with NEDD4L and mimic-NC. The experiment was repeated three times
Fig 5: Hypoxic glioma cell-derived EVs harboring miR-10b-5p facilitate M2 polarization of macrophages, thus increasing glioma development. (A) Heatmap of differential miRNAs in glioma-related miRNA expression microarray. The x-axis represents the sample number, the y-axis represents the miRNA name, and the upper right histogram is color scale. (B) Intersection of differential miRNAs with miRNAs in blood EVs in EVmiRNA database. The middle part is the intersection of two sets of data. (C) The expression of miR-10b-5p in brain tissues of 40 cases of glioma and 15 cases of non-glioma examined by RT-qPCR. (D) The expression of miR-10b-5p in N-EV and H-EV determined by RT-qPCR. (E) The expression of miR-10b-5p in U937 cells co-cultured with N-EV and H-EV measured by RT-qPCR. (F) The expression of miR-10b-5p in U937 cells treated with miR-10b-5p-mimic evaluated by RT-qPCR. (G) The mRNA expression of iNOS, TNF-a, Arg-1, and IL-10 in U937 cells treated with miR-10b-5p-mimic assessed by RT-qPCR. (H) Immunoblotting of protein expression of iNOS and Arg-1 in U937 cells treated with miR-10b-5p-mimic. (I) The expression of TNF-a and IL-10 in U937 cell supernatant treated with miR-10b-5p-mimic detected by ELISA. (J) The proportion of CD11b+CD163+ cells in U937 cells treated with miR-10b-5p-mimic evaluated by flow cytometry. (K) The proliferation in glioma U87 and A172 cells co-cultured with miR-10b-5p- mimic or mimic-NC transfected U937 cells tested by EDU. (L) Cell migration in U87 and A172 cells co-cultured with miR-10b-5p-mimic or mimic-NC transfected U937 cells examined by Transwell. (M) Cell invasion in U87 and A172 cells co-cultured with miR-10b-5p-mimic or mimic-NC transfected U937 cells examined by Transwell. *p < 0.05 versus brain tissues from non-glioma or U937 cells treated with PBS or mimic-NC. # p < 0.05 versus U937 treated with normal oxygen-induced glioma-derived EVs. & p < 0.05 versus U87 or A172 co-cultured with U937 cells without any treatment. The experiment was repeated three times
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