Fig. 4.

ERβ induces autophagy and suppresses the migration and invasion of breast cancer cells. a TEM analysis was performed on MDA-MB-231 cells treated or not treated for 24 h with 100 nM DPN. DPN-treated cells displayed several autophagic vacuoles with the characteristic double membrane (white arrowheads) which were not observed in the control (scale bar, 5 μm). b Immunofluorescence analysis showed that the numbers of LC3-II puncta (white arrowheads) were increased after DPN treatment. Nuclei were stained with DAPI (Scale bar, 20 μm). c The expression of LC3B was detected by western blot in DPN-treated or untreated MDA-MB-231 cells. Actin served as a loading control. d The expression of LC3B was detected by western blot in MDA-MB-231 cells cotreated with DPN and CQ (25 μM, 24 h) or DPN and 3-MA (5 mM, 24 h). CQ and 3-MA were pretreated for 2 h before treatment with DPN. Wound healing (e) and Transwell migration (f) assays showed that the DPN-inhibited migration (scale bar, 200 μm) and invasion (scale bar, 50 μm) abilities were attenuated by CQ and 3-MA. CQ and 3-MA were pretreated for 2 h before treatment with DPN. Data are presented as mean ± SD. The data shown are representative results of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001