Fig. 1. Activation of ERβ inhibits cell proliferation while upregulating ZFHX3 but downregulating MYC and CCND1 in both C4-2B and LNCaP cell lines.

a Expression of ZFHX3, AR, ERα, and ERβ in human prostate epithelial cell lines, as determined by Western blotting. Breast cancer cell lines MCF-7 and MDA-MB-231 were used as positive and negative controls, respectively, for ERα. b Treatment with DPN, an ERβ activator, decreased cell proliferation in two-dimensional (2D) culture in both C4-2B (left) and LNCaP (right) cell lines. Cells were serum-starved for 48 h before indicated DPN treatments. Optical densities represent cell numbers. n = 4. c Knockdown of ERβ eliminated the inhibitory effect of DPN on colony formation in both C4-2B (left) and LNCaP (right) cell lines in plates pre-coated with 0.35% soft agar. Cells were transfected with siESR2-2, which showed the highest efficiency of knockdown among the three siRNAs against ESR2, for 24 h and treated with DPN (0.1 µM) for 2 weeks. Colonies with a diameter > 100 µm were counted. Knockdown of ERβ was validated by Western blotting. n = 3. d Inhibition of ERβ function by its antagonist PHTPP eliminated the inhibitory effect of DPN on colony formation. n = 3. e DPN upregulates ZFHX3 expression but downregulates MYC and CCND1 in C4-2B and/or LNCaP cells. DPN treatments were at 0, 0.01, 0.1, and 1 µM for 48 h, and protein expression was determined by Western blotting. f Inhibition of ERβ function by its antagonist PHTPP eliminated the inhibitory effect of DPN on MYC expression in C4-2B cells. DPN and PHTPP were at 0.5 μM. *P < 0.05; **P < 0.01; ns, not significant. ZFHX3, zinc-finger homeobox 3; AR, androgen receptor; ERα, estrogen receptor alpha; DPN, diarylpropionitrile; siRNA, small interfering RNA; PHTPP, 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol