Fig. 5

GM-CSF-initiated STAT5 signaling enhances AHR stability by inhibiting AHR ubiquitination. a Western blotting analysis of pSTAT5 and STAT5 expressions in peritoneal macrophages treated with serum from mice inoculated with or without 4T1 cells for 14 days (repeated twice). b Western blotting analysis of AHR, pSTAT5, and STAT5 levels in peritoneal macrophages treated with 4T1-CM at indicated time points (repeated three times). c Western blotting analysis of pSTAT5 and STAT5 expressions in peritoneal macrophages treated with CM from control 4T1 cells or Csf2 knockout 4T1 cells at indicated time points. d Western blotting analysis of AHR, pSTAT5, and STAT5 expressions in peritoneal macrophages treated with GM-CSF (0, 10, 20 ng/mL) for 30 min, 60 min, and 120 min. e Western blotting analysis of AHR expression in STAT5-IN-1-treated peritoneal macrophages, with or without 4T1-CM treatment for 24 h (repeated twice). f Western blotting analysis of AHR expression in peritoneal macrophages treated with GM-CSF (10 ng/mL or 5 ng/mL), with or without the addition of STAT5-IN-1 (10 or 20 μM) (repeated twice). g Histogram of PD-L1 expression on peritoneal macrophages treated with DMSO or GM-CSF (10 ng/mL) for 36 h, with or without the addition of STAT5-IN-1 (10 or 20 μM) (n ≥ 4 for each group, repeated twice). The bar graph shows MFI of PD-L1 on each group of macrophages. h mRNA levels of Ahr in peritoneal macrophages treated with or without 4T1-CM, in the presence or absence of STAT5-IN-1 (1, 5, or 10 μM) for 24 h (n ≥ 4 for each group, repeated three times). i The protein levels of AHR in peritoneal macrophages treated with 4T1-CM and CHX (5 µg/ml), with or without the addition of STAT5-IN-1 (10 μM) (repeated three times). j The protein levels and quantification of AHR in peritoneal macrophages treated with 4T1-CM and MG132 (5 μM) for 6 or 8 h, with or without the addition of STAT5-IN-1 (10 μM). k Western blotting analysis of ubiquitin in macrophages treated with 4T1-CM and STAT5-IN-1. Peritoneal macrophages cultured in 4T1-CM were treated with DMSO or STAT5-IN-1 (10 μM) for 12 h. The cell lysates were immunoprecipitated with an anti-AHR antibody and immunoblotted with an anti-ubiquitin antibody (repeated three times). Data are analyzed by unpaired two-tailed t-test (g, h, j) and presented as mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001, ns no significance