Fig 1: AHR and GPER are involved in the activation of the EGFR/ERK1/2/c-Fos transduction pathway by 3MC. Phosphorylation of EGFR (a, c) and ERK1/2 (b, d) in SkBr3 cells and CAFs treated for 15 min with vehicle (-) or 1 µM 3MC alone or in combination with either 1 µM AHR inhibitor CH223191 or 100 nM GPER antagonist G15, as indicated. EGFR and ERK2 serve as loading controls for pEGFR and pERK1/2, respectively. Results shown are representative of three independent experiments. e 1 µM 3MC induces the mRNA expression of c-Fos in SkBr3 cells and CAFs, as indicated. Data obtained by real-time PCR in three independent experiments performed each in triplicate were normalized to 18S expression and shown as fold changes of c-Fos expression upon treatment with 3MC respect to cells treated with vehicle (-). (¦) indicates P < 0.05 for cells receiving treatments versus vehicle (-). Evaluation of c-Fos protein levels in SkBr3 cells (f) and CAFs (g) upon a 6 h treatment with vehicle (-) and 1 µM 3MC alone or in combination with either 1 µM AHR inhibitor CH223191 or 100 nM GPER antagonist G15. ß-actin serves as a loading control. h-j Co-immunoprecipitation studies performed in SkBr3 cells treated with vehicle (-) or 1 µM 3MC for 1 h, as indicated. Cell lysates were immunoprecipitated with either anti-GPER (h), or anti-AHR (i) or anti-EGFR (j) antibodies. Immunocomplexes were analyzed by immunoblot with antibodies against the indicated proteins. In control samples, nonspecific IgG was used instead of the primary antibody. k Total lysates (input) were evaluated as control. Results shown are representative of at least two independent experiments
Fig 2: Schematic representation of CYP1B1 regulation by both AHR and GPER signaling
Fig 3: AHR nuclear translocation induced by 3MC is prevented by the AHR inhibitor CH223191 and the GPER antagonist G15. The AHR nuclear translocation in SkBr3 cells exposed for 1 h to 1 µM 3MC is prevented by 1 µM AHR inhibitor CH223191 and 100 nM GPER antagonist G15. Merge panels depict the overlap of the two fluorophores used to assess the nuclear translocation of AHR. Red signal: AHR. Blue signal: Nuclei. Images shown are representative of ten random fields from three independent experiments. Scale bar 12.06 µm
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. ß-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. ß-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 (-)
Fig 5: AHR and GPER are involved in the up-regulation of cyclin D1 by 3MC. a Cyclin D1 mRNA expression in SkBr3 cells and CAFs treated with vehicle (-) and 1 µM 3MC, as indicated. mRNA expression of cyclin D1 in SkBr3 cells (b) and CAFs (d) upon treatments 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. Data obtained by real-time PCR in three independent experiments performed each in triplicate were normalized to 18S expression and shown as fold changes of Cyclin D1 expression induced by treatments with respect to cells treated with vehicle (-). Cyclin D1 protein levels in SkBr3 cells (c) and CAFs (e) upon treatments 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. Cyclin D1 protein levels in SkBr3 cells (f) and CAFs (j) upon treatments for 18 h with vehicle (-) and 1 µM 3MC alone or in combination with 5 µM CYP1B1 activity inhibitor TMS. Cyclin D1 protein levels in SkBr3 cells (g) and CAFs (k) transiently transfected with shRNA or shCYP1B1 for 24 h and then treated for 18 h with vehicle (-) and 1 µM 3MC. h, l Efficacy of CYP1B1 silencing. Cyclin D1 protein levels in SkBr3 cells (i) and CAFs (m) treated for 18 h with vehicle (-) and 1 µM 3MC alone or in combination with 100 nM SP1 inhibitor Mithramycin A (MTM A). ß-actin serves as a loading control. Results shown are representative of three independent experiments. (¦) P < 0.05 for cells receiving treatments versus vehicle (-)
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