Xenbase Image ID: 172387

Figure 5. ERK3 knockdown induces thickened epithelia with defective adherens and tight junctions in MCF7 cells. A, Two different siRNAs targeting human ERK3 were effective in MCF7 cells. Cells were transfected with 20 nM control siRNA, ERK3 siRNA1 or ERK3 siRNA2, cultured for 3 days, and then subjected to real-time quantitative RT-PCR analyses. The expression levels of human ERK3 were normalized to those of human GAPDH. Shown are all data points from three or seven independent experiments. The bars represent the average±S.D. The normalized ERK3 expression level in control cells was defined as 1.0 in each experiment. **P<0.01 by Dunnett test. B, Typical morphologies of MCF7 cells 3 days after transfection with the indicated siRNAs (20 nM). The data are representative of three images from three independent experiments. Scale bars, 100 m. C, Proliferation was assessed by cell counting. MCF7 cells (3.5 x 105) were transfected with the indicated siRNAs (20 nM) and counted 3 days after transfection. Shown are all data points from three independent experiments. The bars represent the average±S.D. n.s., not significant by Dunnett test. D-H, MCF7 cells were transfected with the indicated siRNAs (20 nM), fixed 3 days after transfection, and subjected to triple staining with Hoechst (blue), Alexa Fluor 568 phalloidin (red), and an antibody against E- cadherin (D, E) or ZO-1 (F, G) (green). 3D images obtained by confocal microscopy were viewed from the apical (D, F), lateral (E) or apico-lateral (G) sides. D, E, Shown are representative images of 6-10 3D images from three independent experiments. F, G, Shown are representative images of 6-13 3D images from three independent experiments. D, F, Higher magnification images of the regions outlined in white in the upper panels are shown in the lower panels. Images of Alexa Fluor 568 phalloidin staining are not shown. Scale bars, 20 μm. E, Scale bars, 10 μm. H, The scatter plot represents the maximum thickness of the cell layers (or colonies) in the 3D images in D-G. The bars represent the average±S.D. *P<0.05, **P<0.01 by Dunnett test. Image published in: Takahashi C et al. (2018) Copyright © 2018. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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