Lappano R , Rosano C , Pisano A , Santolla MF , De Francesco EM , De Marco P , Dolce V , Ponassi M , Felli L , Cafeo G , Kohnke FH , Abonante S , Maggiolini M .

	Fig. 1. Chemical structure of meso-octamethylcalix[4]pyrrole (C4PY).
	Fig. 2. Ligand binding modes to GPER. (A) C4PY in the protein binding cleft is drawn in green. The protein surface is colored according to its electrostatic potential (blue positive, red negative). The same ligand binding mode is schematically reported in panel B, where the interacting amino acids are indicated as dark gray sticks. (C,D) The agonists GPER-L1 and GPER-L2 are drawn in light green (C) and purple (D) sticks, respectively. Binding mode of G-1 (cyan) is shown in panel E and the full-antagonist MIBE (orange) in panel F.
	Fig. 3. C4PY is a ligand of GPER. (A) C4PY competes with [3H]E2 for binding to GPER in SkBr3 cells. Competition curves of increasing concentration of unlabeled E2, G-1 and C4PY expressed as a percentage of maximum specific [3H]E2 binding. Each data point represents the mean±s.d. of triplicate samples of three separate experiments. (B) C4PY competes with [5,6-3H] nicotinic acid (NA) for binding to GPER in SkBr3 cells. Competition curves of increasing concentration of unlabeled NA, G-1 and C4PY expressed as a percentage of maximum specific [5,6-3H] NA binding. Each data point represents the mean±s.d. of three separate experiments performed in triplicate.
	Fig. 4. C4PY exerts inhibitory effects through GPER in SkBr3 breast cancer cells. (A) ERK1/2 and Akt activation in SkBr3 cells treated for 15 min with 100 nM E2 or 1 µM G-1 is prevented in the presence of 1 µM C4PY. (B) Densitometric analysis of the blots normalized to ERK2 and Akt, respectively. Each data point represents the mean±s.d. of three independent experiments. (C) The mRNA expression of fos and EGR1 induced in SkBr3 cells by 1 h treatment with 100 nM E2 and 1 µM G-1 is inhibited in the presence of 1 µM C4PY, as evaluated by real-time PCR. Results obtained from experiments performed in triplicate were normalized for 18S expression and shown as fold change of RNA expression compared to cells treated with vehicle. Each data point represents the mean±s.d. of three independent experiments performed in triplicate. (D) The transactivation of fos and EGR1 luciferase reporter genes transfected in SkBr3 cells induced by 100 nM E2 and 1 µM G-1 is inhibited by 1 µM C4PY. Luciferase activity was normalized to the internal transfection control Renilla luciferase; values are presented as fold change (mean±s.d.) of vehicle control and represent three independent experiments, each performed in triplicate. (E) The proliferation of SkBr3 cells upon treatment with 100 nM E2 and 100 nM G-1 is inhibited by 1 µM C4PY, as indicated. Cells were treated for 5 days with the indicated treatments and counted on day 6. Proliferation of cells receiving vehicle was set as 100%, upon which cell growth induced by treatments was calculated. Each data point is the average ±s.d. of three independent experiments performed in triplicate. (•) indicates P<0.05 for cells receiving vehicle (–) versus treatments.
	Fig. 5. C4PY exerts inhibitory effects through GPER in CAFs. (A) ERK1/2 and Akt activation in CAFs treated for 5 min with 1 nM E2 and 100 nM G-1 is prevented by 1 µM C4PY. (B) Densitometric analysis of the blots normalized to ERK2 and Akt, respectively. Each data point represents the mean±s.d. of three independent experiments. (C) The mRNA expression of CTGF and Cyr61 induced in CAFs by 1 h treatment with 1 nM E2 and 100 nM G-1 is prevented by 1 µM C4PY, as evaluated by real-time PCR. Results obtained from experiments performed in triplicate were normalized for 18S expression and shown as fold change of RNA expression compared to cells treated with vehicle. Each data point represents the mean±s.d. of three independent experiments performed in triplicate. (D) CTGF and Cyr61 protein expression induced in CAFs by 2 h treatment with 1 nM E2 and 100 nM G-1 is inhibited in the presence of 1 µM C4PY. (E) Densitometric analyses of the blots normalized to β-actin; values shown represent the mean±s.d. of three independent experiments. (F) The migration of CAFs upon treatment with 1 nM E2 and 100 nM G-1 is inhibited by 1 µM C4PY, as evaluated by Boyden Chamber assay. Each data point is the average ±s.d. of three independent experiments performed in triplicate. (•) and (◦) indicate P<0.05 for cells receiving vehicle (–) versus treatments.
	Fig. 6. The migration of CAFs induced by E2 (1 nM) and G-1 (100 nM) is inhibited by 1 µM C4PY, as determined by wound-healing assay. Data are representative of three independent experiments performed in triplicate.
	Fig. 7. C4PY does not interfere with the ER-mediated signaling. (A) MCF-7 cells were transfected with an ER luciferase reporter gene along with the internal transfection control Renilla luciferase and then treated with 10 nM E2 in combination with 1 µM C4PY or ICI, as indicated. The normalized luciferase activity values of cells treated with vehicle were set as 1-fold induction, upon which the activity induced by treatments was calculated. Each data point represents the mean±s.d. of three experiments performed in triplicate. (B) The mRNA expression of cyclin D1 (Cyc D1), progesterone receptor (PR) and pS2 induced in MCF-7 cells by 24 h treatment with 10 nM E2 is inhibited by 1 µM ICI, but not by 1 µM C4PY, as evaluated by real-time PCR. Results obtained from experiments performed in triplicate were normalized for 18S expression and shown as fold change of RNA expression compared to cells treated with vehicle. Each data point represents the mean±s.d. of three independent experiments performed in triplicate. (C) The proliferation of MCF-7 cells upon treatment with 10 nM E2 is inhibited by 1 µM ICI, but not by 1 µM C4PY, as indicated. Cells were treated for 5 days with the indicated treatments and counted on day 6. Proliferation of cells receiving vehicle was set as 100%, upon which cell growth induced by treatments was calculated. Each data point is the average ±s.d. of three independent experiments performed in triplicate. (•) indicates P<0.05 for cells receiving vehicle (–) versus treatments.

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