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. 2020 Jan 25;12(2):289. doi: 10.3390/cancers12020289

Figure 4.

Figure 4

GPER activation inhibits epithelial–mesenchymal transition in Suit2-007 cells. (a) Left: Immunofluorescence images of Suit2-007 cells stained for β-catenin, DAPI and actin, for control and 1 μM G1 (GPER agonist) after 24 h of culture. White arrow indicates nuclear localisation of β-catenin, orange arrow indicates cytoplasmic localisation of β-catenin. Scale bar = 25 μm. Right: Quantification of β-catenin localisation. For Suit2-007, control and G1, n = 18 and 18 regions of interest respectively. For PC-3, control and G1, n = 17 and 16 regions of interest respectively. Mann–Whitney test for statistical significance, * p < 0.05, *** p < 0.001. (b) Left: Immunofluorescence images of Suit2-007 cells stained for vimentin, for control and G1 (1 µM) after 24 h of culture. Scale bar = 25 μm. Right: Quantification of vimentin immunofluorescence images respectively. For control and G1, n = 30 and 30 regions of interest respectively. Mann–Whitney test for statistical significance, * p < 0.05, *** p < 0.001. (c) Immunofluorescence images of Suit2-007 cells stained for β-catenin, DAPI and actin, for control, 5 µM tamoxifen, 5 µM tamoxifen + 1 µM ICI 182780 (ER antagonist), 5 µM tamoxifen + 2 µM G15 (GPER antagonist) for 72 h of culture. White arrows indicate nuclear localisation of β-catenin, orange arrows indicate cytoplasmic localisation of β-catenin. Scale bar = 25 μm. (d) Quantification of β-catenin localisation. For Suit2-007, control, 5 µM tamoxifen, 5 µM tamoxifen + 1 µM ER Ant, 5 µM tamoxifen + 2 µM GPER Ant (all for 72 h), n = 15, 11, 15, 18 regions of interest respectively. For PC-3, control, 5 µM tamoxifen, 5 µM tamoxifen + 1 µM ER Ant, 5 µM tamoxifen + GPER Ant (all for 72 h), n = 16, 15, 10, 11 regions of interest respectively. Mann–Whitney test for statistical significance, * p < 0.05, ** p < 0.01, *** p < 0.001. (e) Immunofluorescence images of Suit2-007 cells stained for vimentin, for control, 5 µM tamoxifen, 5 µM tamoxifen + 1 µM ER Ant, 5 µM tamoxifen + 2 µM GPER Ant for 72 h of culture. (f) Quantification of immunofluorescence intensity for vimentin. For Suit2-007, control, 5 µM tamoxifen, 5 µM tamoxifen + 1 µM ER Ant, 5 µM tamoxifen + GPER Ant (all for 72 h), n = 16, 10, 10, 17 regions of interest respectively. For PC-3, control, 5 µM tamoxifen, 5 µM tamoxifen + 1 µM ER Ant, 5 µM tamoxifen + 2 µM GPER Ant (all for 72 h), n = 17, 15, 16, 15 cells regions of interest respectively. Mann–Whitney test for statistical significance, * p < 0.05, ** p < 0.01, *** p < 0.001. (g) Quantitative PCR analysis of the YAP-target genes for vimentin and E-cadherin, normalised to expression of RPLP0. For Mann–Whitney test between each condition and G1 condition, ** p < 0.01, *** p < 0.001. n = three independent samples. (h) Left: Crystal Violet stained cells on Transwells for control and G1-treated conditions. Right: Count of invaded cells in imaged region for control and G1 (1 µM, 24 h) treated cells. For control and G1, n = 23 and 21 regions respectively. *** represents Mann–Whitney test, p < 0.001. Scale bar = 100 µm.