Fig. 2

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. β-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