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. 2019 Sep 3;55(10):838–853. doi: 10.1007/s11626-019-00403-x

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© The Author(s) 2019

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

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Figure 1. — Phenotypic and functional characterization of D492M and D492HER2 cell lines. (a) D492M and D492HER2 are isogenic EMT-derived sublines of D492, a breast epithelial cell line with progenitor properties, which in 3D cell culture forms branching structures, resembling the TDLUs of the breast. D492M and D492HER2 were generated when D492 underwent endothelial-induced and oncogene (HER2)-induced EMT, respectively. D492M and D492HER2 show similar EMT-phenotype in 2D, but in 3D, D492M generate spindle-like colonies whereas D492HER2 generate spindle-like and grape-like colonies. D492M and D492HER2 also differ in their ability to generate tumors, as only D492HER2 is able to form tumors. Scale bar = 200 μm. (b) D492M and D492HER2 have lost expression of epithelial markers (E-cadherin, cytokeratin-14, and cytokeratin-19) and gained expression of mesenchymal markers (Axl and vimentin) as shown by western blotting and immunofluorescence. Scale bar = 100 μm. (c) D492M and D492HER2 show reduction in the expression of epithelial microRNAs such as miR-200c, miR-203, and miR-205 compared to D492. The lowest levels of expression are found in D492M (mean ± SD, n = 2). (d) In transwell migration and invasion assays, D492HER2 has an increased ability to migrate and invade compared to D492 and D492M. Results are shown as average number of cells per field (mean ± SEM, n = 3). One-way analysis of variance (ANOVA) and Dunnett’s multiple comparison test were used to test significance (*p ≤ 0.05, **p ≤ 0.01). (e) D492HER2 cells proliferate at a higher rate than D492 and D492M cells as shown by staining with crystal violet. Results are shown as average of four replicates (mean ± SD). Statistical significance was assessed using multiple t tests (one per row) (**p ≤ 0.01, ****p ≤ 0.0001). (f) Furthermore, increased proliferation rate of D492HER2 compared to D492 and D492M cells was demonstrated using PrestoBlue™ Cell Viability reagent. Results are shown as average of eight replicates normalized to D492 (mean ± SD). One-way analysis of variance (ANOVA) and Dunnett’s multiple comparison test were used to test significance (****p ≤ 0.0001). (g) In addition, D492HER2 cells are more susceptible to chemically induced apoptosis as compared to D492 and D492M cells. Caspase 3/7 luciferase activity was measured by luminescence and normalized to D492 (mean ± SEM, n = 2). Significance was assessed with one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison test (*p ≤ 0.05).