Fig. 4

GM-CSF released from breast cancer cells promotes AHR expression in macrophages. Quantification of GM-CSF levels in both serum (a) and lung tissue (b) of mice inoculated with 4T1 cells in the mammary gland fat pad for 2 weeks using ELISA (n = 6 for each group). c AHR expression in peritoneal macrophages cultured in normal medium, or CM from control 4T1 cells, or sgCsf2 transfected 4T1 cells for 12 h (repeated twice). d Histogram showing PD-L1 expression on peritoneal macrophages cultured in normal medium, CM from control 4T1 cells, or CM from sgCsf2-transfected 4T1 cells for 24 h (n ≥ 4 for each group, repeated three times). The bar graph shows MFI of PD-L1 on each group of macrophages. e mRNA expression of Ahr in peritoneal macrophages treated with GM-CSF at indicated concentrations for 24 h (n = 3 for each group, repeated twice). f Western blotting analysis and quantification of AHR expression in peritoneal macrophages treated with GM-CSF (10 ng/mL) for 24 h. g Western blotting analysis of AHR expression in the cytoplasm and nucleus of macrophages treated with or without GM-CSF. h Histogram showing PD-L1 expression on WT or Ahr^−/− peritoneal macrophages treated with or without GM-CSF (2.5, 5, or 10 ng/mL) for 12 h. The bar graph shows MFI of PD-L1 on each group of macrophages (n ≥ 4 for each group). Data are analyzed by unpaired two-tailed t-test (b, d, e, h) or Mann Whitney test (a) or paired two-tailed t-test (f) and presented as mean ± SEM (a, b, d, e, h) or symbols & lines (f). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001