Fig 2: Effects of AhR Antagonists on AhR, COX-2, RANKL, and PGE2, CXCR4, CXCL12 expressions. MG-63 cells were cultured for 72 h with toluene (vehicle control), 10 nM TCDD and 10 nM TCDD plus CH-223191 for 72 h. (A) AhR, COX-2 and RANKL protein levels were measured by western blot analysis. (B) AhR protein levels at 72 h. (C) COX-2 protein levels at 72 h. (D) RANKL protein levels at 72 h. Data are presented as the mean ± standard error of the mean (n=3). *P<0.05, **P<0.01 and ***P<0.005 vs. the toluene-treated control (n=3). CH, 10 µM CH-223191; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; COX-2, cyclooxygenase-2; RANKL, receptor activator of nuclear factor-?B ligand.

Fig 3: PGE2, CXCR4, CXCL12 profiles derived from MG-63 cells treated with AhR agonists. (A) Cells were treated with toluene (vehicle control), 1 or 10 nM TCDD and 10 nM TCDD plus CH-223191 for 72 h. PGE2 protein levels in the supernatants were measured by ELISA. (B) CXCR4 receptor expressions were measured by flow cytometry after toluene (vehicle control), 1 nM, 10 nM TCDD and 10 nM TCDD plus CH-223191 treatment for 24 h. The number represented the percentage of total cells. (C) CXCL12 protein production in the supernatant after cells were treated with toluene (vehicle control), 1 nM, 10 nM TCDD and 10 nM TCDD plus CH-223191 for 24 h was measured by ELISA. Data are presented as the mean ± standard error of the mean (n=3). **P<0.01 vs. the toluene-treated control (n=3). CH, 10 µM CH-223191; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; AhR, aryl hydrocarbon receptor; PGE2, prostaglandin E2.

Fig 4: Biomarker immunocytochemistry in MCF-7 and MDA-MB-231 cells following PEDF treatment in pre- versus post-menopausal oestrogens. Biomarker expression via immunocytochemistry staining (blue) in MCF-7 and MDA-MB-231 cells following incubation in media supplemented with either pre-menopausal (E1: 258 pM; E1S: 4130 pM; E2: 271 pM) or post-menopausal (E1: 72 pM; E1S: 541 pM; E2: 14.6 pM) oestrogen levels and treated with recombinant PEDF (100 nM) or control for 24-h. CXCR4 staining under pre-menopausal conditions in MCF-7 treated with control (A) or PEDF (B) and in MDA-MB-231 treated with control (E) or PEDF (F), or under post-menopausal conditions in MCF-7 treated with control (C) or PEDF (D) and in MDA-MB-231 treated with control (G) or PEDF (H). Phosphorylated (p-)NF?B-p65 staining under pre-menopausal conditions in MCF-7 treated with control (I) or PEDF (J) and in MDA-MB-231 treated with control (M) or PEDF (N), or under post-menopausal conditions in MCF-7 treated with control (K) or PEDF (L) and in MDA-MB-231 treated with control (O) or PEDF (P). TNF-a staining under pre-menopausal conditions in MCF-7 treated with control (Q) or PEDF (R) and in MDA-MB-231 treated with control (U) or PEDF (V), or under post-menopausal conditions in MCF-7 treated with control (S) or PEDF (T) and in MDA-MB-231 treated with control (W) or PEDF (X). uPAR staining under pre-menopausal conditions in MCF-7 treated with control (Y) or PEDF (Z) and in MDA-MB-231 treated with control (AC) or PEDF (AD), or under post-menopausal conditions in MCF-7 treated with control (AA) or PEDF (AB) and in MDA-MB-231 treated with control (AE) or PEDF (AF). Biomarker staining intensity in cells treated with control or PEDF for CXCR4 (a), p-NF?B-p65 (c), TNF-a (e), and uPAR (g). Biomarker fold change in PEDF-treated cells compared to control in pre- or post-menopausal conditions for CXCR4 (b), p-NF?B-p65 (d), TNF-a (f), and uPAR (h). Scale bar 50 µM. **** p < 0.0001; *** p = 0.0001–0.0009; ** p = 0.001–0.009; * p = 0.01–0.05.

Fig 5: Biomarker expression following PEDF and JSH-23 treatment in MCF-7 and MDA-MB-231 cells in pre- and post-menopausal conditions. Biomarker expression via immunocytochemistry staining (blue) in cells following incubation in pre- (E1: 258 pM; E1S: 4130 pM; E2: 271 pM) or post-menopausal (E1: 72 pM; E1S: 541 pM; E2: 14.6 pM) levels of oestrogens and treatment with PEDF (100 nM), JSH-23 (20 µM), PEDF (100 nM) + JSH-23 (20 µM), or control for 24-h. Phosphorylated (p-)NFκB-p65 expression under pre-menopausal conditions in MCF-7 treated with control (A), PEDF (B), JSH-23 (C), or PEDF + JSH-23 (D) and in MDA-MB-231 treated with control (I), PEDF (J), JSH-23 (K), or PEDF + JSH-23 (L), and under post-menopausal conditions in MCF-7 treated with control (E), PEDF (F), JSH-23 (G), or PEDF + JSH-23 (H) and in MDA-MB-231 treated with control (M), PEDF (N), JSH-23 (O), or PEDF + JSH-23 (P). TNF-α expression under pre-menopausal conditions in MCF-7 treated with control (Q), PEDF (R), JSH-23 (S), or PEDF + JSH-23 (T) and in MDA-MB-231 treated with control (Y), PEDF (Z), JSH-23 (AA), or PEDF + JSH-23 (AB), or under post-menopausal conditions in MCF-7 treated with control (U), PEDF (V), JSH-23 (W), or PEDF + JSH-23 (X) and in MDA-MB-231 treated with control (AC), PEDF (AD), JSH-23 (AE), or PEDF + JSH-23 (AF). CXCR4 expression under pre-menopausal conditions in MCF-7 treated with control (AG), PEDF (AH), JSH-23 (AI), or PEDF + JSH-23 (AJ) and in MDA-MB-231 treated with control (AO), PEDF (AP), JSH-23 (AQ), or PEDF + JSH-23 (AR) or under post-menopausal conditions in MCF- treated with control (AK), PEDF (AL), JSH-23 (AM), or PEDF + JSH-23 (AN) and in MDA-MB-231 treated with control (AS), PEDF (AT), JSH-23 (AU), or PEDF + JSH-23 (AV). uPAR expression under pre-menopausal conditions in MCF-7 treated with control (AW), PEDF (AX), JSH-23 (AY), or PEDF + JSH-23 (AZ) and in MDA-MB-231 treated with control (BE), PEDF (BF), JSH-23 (BG), or PEDF + JSH-23 (BH) or under post-menopausal conditions in MCF-7 treated with control (BA), PEDF (BB), JSH-23 (BC), or PEDF + JSH-23 (BD) and in MDA-MB-231 treated with control (BI), PEDF (BJ), JSH-23 (BK), or PEDF + JSH-23 (BL). Biomarker staining intensity in cells treated with control, PEDF, JSH-23, or PEDF + JSH-23 for p-NFκB-p65 (a) TNF-α (c), uPAR (e), and CXCR4 (g). Biomarker fold change in PEDF-treated cells versus control in pre- or post-menopausal conditions for p-NFκB-p65 (b), TNF-α (d), uPAR (f), and CXCR4 (h). Scale bar 50 µM.