Fig. 6

Transduction pathways involved in the proliferative effects triggered by 3MC. The proliferation of SkBr3 cells (a) and CAFs (e) induced by 1 μM 3MC is prevented by 1 μM AHR inhibitor CH223191, 100 nM GPER antagonist G15, 5 μM CYP1B1 inhibitor TMS and 100 nM SP1 antagonist mithramycin A (MTM A). The growth effects induced by 1 μM 3MC in SkBr3 cells (b) and CAFs (f) are prevented silencing either CYP1B1 or GPER expression. Cells were transfected every 2 days with shRNA, shCYP1B1 or shGPER, treated every day with ligands and then counted on day 5. Efficacy of the silencing of CYP1B1 (c, g) and GPER (d, h). β-actin serves as a loading control. Proliferation of cells treated with vehicle (−) was set as 100% upon which cell growth induced by treatments was calculated. Each data point is the mean ± SD of three independent experiments performed in triplicate. i Representative images of SkBr3 spheroids (a single spheroid per well) grown on agar-coated plates after 20 days treatment with vehicle (−) or 1 μM 3MC alone or in combination with 1 μM AHR inhibitor CH223191, 100 nM GPER antagonist G15, 5 μM CYP1B1 inhibitor TMS and 100 nM SP1 antagonist MTM A, as indicated. j Evaluation of SkBr3 cell growth upon treatments, as indicated, vehicle (−) was set as 100% upon which the results induced by treatments was calculated. Each column represents the mean ± SD of two independent experiments, each performed in triplicate. (■) indicates P < 0.05 for cells receiving treatments versus vehicle (−)