Fig 1: Concentration of pro-inflammatory cytokines by ELISA in the serum samples of patients with MDD (n = 10) and healthy controls (n = 10). Concentrations of (A) IL-1ß; (B) IL-2; (C) IL-6; (D) TNF-a; (E) IFN-?; and (F) G-CSF. The concentration of pro-inflammatory cytokines in the serum of MDD patients was significantly increased. The experiments were repeated three times. The results are expressed as mean ± SD. *p < 0.05 vs. control group. MDD: Major depressive disorder; IL: Interleukin; TNF-a: Tumor necrosis factor alpha; IFN-?: Interferon gamma; G-CSF: Granulocyte colony-stimulating factor; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; NF-?B: Nuclear factor kappa-light-chain-enhancer of activated B cells; ELISA: Enzyme-linked immunosorbent assay.
Fig 2: Exosomes secreted from M1 macrophages harboring miR-16-5p promote the activation of T cells in the co-culture system with GC cells. (A) Flow cytometry of PD-L1 expression in GC cells treated with exo-miR-16-5p mimic and exo-miR-16-5p inhibitor. (B) Flow cytometry of CD3+ T cell proliferation in GC cells co-cultured with T cells for 24 h after treated with exo-miR-16-5p mimic or exo-miR-16-5p inhibitor. (C) Flow cytometry of the number of INF-?+ T cells in GC cells co-cultured with T cells for 24 h after treated with exo-miR-16-5p inhibitor or exo-miR-16-5p inhibitor. (D) ELISA was performed to determine levels of IL-2, TNF-a and INF-? in the supernatant of T cells after treated with exosomes. (E) Representative images of tumors in mice after tail injection of GC cells stably transfected with exo-miR-16-5p mimic or exo-miR-16-5p inhibitor as well as quantification of tumor volume and weight (n = 5). *p < 0.05 cells treated with exo-mimic-NC; #p < 0.05 compared with cells treated with exo-inhibitor-NC. Measurement data representative of three independently conducted experiments expressed as the mean ± standard deviation. Comparisons between two groups are analyzed by non-paired t test.
Fig 3: MACC1 regulates immune cell-mediated gastric cancer killing and cytokine concentrations in vitro. (A) Silencing of MACC1 induces the immune cell-mediated killing of gastric cancer cells. (B) Overexpression of MACC1 inhibits the immune cell-mediated gastric cancer killing. (C) PDL1 inhibition is involved in the regulation of MACC1-mediated immune killing. (D) ELISA analysis of IFN-?, IL-2, IL-10, and TNF-a concentrations in cell supernatants. Data are the mean ± SD (n = 3), and each bar represents the mean of three independent experiments carried out in triplicate. *P < .05 and # P < .05, compared with shMACC1-NC or vector-NC group
Fig 4: Exosomes derived from M1 macrophages promote the immune response of T cells. Mice are treated with PBS or M1 macrophage-derived exosomes. (A) Immunohistochemistry of CD3+ T infiltration in the tumors of mice (400×). (B) Flow cytometry of INF-?+ T cells in CD3+ T cells (n = 5). (C) ELISA of IL-2, TNF-a and INF-? expression in the supernatant of T cells (n = 5). (D) Flow cytometry of PD-1+ T cell proliferation in GC cells treated with M1 macrophage-derived exosomes and anti-PD-L1 after co-cultured with PD-1+ or PD-1– T cells. (E) Flow cytometry of PD-1– T cell proliferation in GC cells treated with M1 macrophage-derived exosomes and anti-PD-L1 after co-cultured with PD-1– T cells or PD-1– T cells. *p < 0.05 compared to mice treated with PBS as well as in comparison to the GC cells without any treatment; #p < 0.05 compared with GC cells treated with M1 macrophage-secreted exosomes. Measurement data representative of three independently conducted experiments expressed as mean ± standard deviation. Comparisons between two groups analyzed by non-paired t test as well as comparisons among multiple groups are analyzed by one-way ANOVA. Post hoc test was conducted using Tukey’s test.
Fig 5: MACC1 regulates immune killing in vivo. (A) qRT-PCR analysis of IFN-?, IL-2, and IL-10 expression in tumor tissue samples. (B) ELISA analysis of IFN-?, IL-2, and TNF-a concentrations in serum samples. Data are the mean ± SD (n = 4), and each bar represents the mean of three independent experiments carried out in triplicate. *P < .05 and #P < .05, compared with shMACC1-NC or vector-NC group
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