Fig. 4

AHR and GPER are involved in CYP1B1 induction by 3MC. a 1 μM 3MC induces the mRNA expression of CYP1B1 in SkBr3 breast cancer cells and CAFs, as indicated. Data obtained by real-time PCR in three independent experiments performed in triplicate were normalized to the expression of 18S and shown as fold changes of CYP1B1 expression upon treatment with 3MC with respect to cells treated with vehicle (−). Evaluation of CYP1B1 protein levels in SkBr3 cells (b) and CAFs (e) upon treatment for 6 h with vehicle (−), 1 μM 3MC alone and in combination with either 1 μM AHR inhibitor CH223191 or 100 nM GPER antagonist G15. The up-regulation of CYP1B1 protein levels induced by a 6 h treatment with 1 μM 3MC is abrogated in SkBr3 cells (c) and CAFs (f) transfected for 24 h with shGPER. d, g Efficacy of GPER silencing. β-actin serves as a loading control. Results shown are representative of three independent experiments. h Luciferase activities of CYP1B1 promoter constructs in SkBr3 cells and CAFs treated for 18 h with vehicle (−) and 1 μM 3MC alone and in combination with either 1 μM AHR inhibitor CH223191 or 100 nM GPER antagonist G15, as indicated. CYP1B1 protein levels in SkBr3 cells (i) and CAFs (j) transfected for 18 h with a vector or DN/c-Fos construct and then treated for 6 h with vehicle (−) and 1 μM 3MC, as indicated. β-actin serves as a loading control. Results shown are representative of three independent experiments. k Luciferase activities of CYP1B1 promoter plasmids in SkBr3 cells and CAFs transfected for 8 h with CYP1B1 constructs, a vector or DN/c-Fos construct and then treated for 18 h with vehicle (−) and 1 μM 3MC. The luciferase activities were normalized to the internal transfection control and values of cells receiving vehicle (−) were set as 1-fold induction upon which the activities induced by treatments were calculated. Each column represents the mean ± SD of three independent experiments, each performed in triplicate. (■) indicates P < 0.05 for cells receiving treatments versus vehicle (−)