Journal: International Journal of Molecular Sciences
Article Title: Antioxidant N-Acetylcysteine Facilitates Breast Cancer Metas-Tasis via Immunosuppressive Reprogramming of Neutrophils
doi: 10.3390/ijms27010526
Figure Lengend Snippet: NAC reprograms neutrophils to be immunosuppressive. ( A ) BM-derived neutrophils were isolated from naïve mice using anti-Ly6G microbeads and treated with vehicle or different concentrations of NAC (0.1–10 µM) for 12 h and then harvested for RNA extraction. Total mRNA was transcribed to cDNA, and the expression of indicated genes was measured by RT-qPCR ( n = 5). ( B – D ) As depicted in the schematic ( B ), BM-derived neutrophils were pre-treated with vehicle or 10 μM NAC and then cultured in RPMI-1640 medium for 12 h. The conditioned medium (CM) derived from vehicle-pre-treated or NAC-pre-treated neutrophils was collected and incubated with splenic T cells in the presence of plate-coated anti-CD3 and soluble anti-CD28, and 5 h later, T cells were harvested to measure the frequencies of CD25 + CD8 + T cells and CD69 + CD8 + T cells ( C ). Then, 72 h later, T cells were subjected to intracellular cytokine staining to measure the frequencies of GZMB + , IFNγ + , and PRF1 + CD8 + T cells ( D ) with flow cytometry ( n = 3 in ( C ); n = 4 in ( D )). ( E – G ) As depicted in the schematic ( E ), E0771 tumor-bearing mice were IP-injected with vehicle or NAC (150 mg/kg) every 2 days from day 7 to day 25. On day 26, lung tissues were harvested to generate single-cell suspensions. Then, neutrophils were sorted from crude lung cells and used to analyze the expression of indicated genes by RT-qPCR ( F ) ( n = 4). Lung Ly6G − cells were incubated with phorbol myristate acetate (PMA) (50 ng/mL, TargetMol), ionomycin (1 µg/mL, TargetMol), GolgiStop (1:1000, BD Biosciences), and GolgiPlug (1:1000, BD Biosciences) for 4 h at 37 °C. The frequencies of GZMB + , IFNγ + , and PRF1 + CD8 + T cells in the lung were measured with flow cytometry ( G ) ( n = 4). n represents the number of biological replicates. Data are shown as mean ± SEM, and statistical significance was determined by one-way ANOVA ( A ) or unpaired Student’s t test ( C , D , F , G ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; NS, not significant.
Article Snippet: Briefly, neutrophils were depleted from lung crude cells with anti-Ly6G MicroBeads to avoid potential artifacts, and lung Ly6G − cells were incubated with PMA (50 ng/mL, TargetMol, Boston, MA, USA), ionomycin (1 μg/mL, TargetMol, Boston, MA, USA), GolgiStop (1:1000, BD Biosciences, San Jose, CA, USA), and GolgiPlug (1:1000, BD Biosciences, San Jose, CA, USA) for 4 h at 37 °C.
Techniques: Derivative Assay, Isolation, RNA Extraction, Expressing, Quantitative RT-PCR, Cell Culture, Incubation, Staining, Flow Cytometry, Injection, Single Cell
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