Fig. 6.

Expression of mutant HRAS^Q61R results in the acquisition of a partial myoepithelial phenotype in non-malignant breast epithelial cells. a Representative western blot (left) analysis of total protein expression of alpha-smooth muscle actin (αSMA), TIMP1, cytokeratin 5 (CK5), E-cadherin, vimentin, and nuclear protein expression of ∆N-p63 and TA-p63 in MCF-10A^P, MCF-10A^H1047R, and MCF-12A cells stably expressing empty vector (EV), HRAS^WT, or mutant HRAS^Q61R. α-Tubulin and Histone H3 were used as protein loading controls for total and nuclear protein expression, respectively. Quantification (right) using LI-COR is shown based on experiments done in triplicate. Comparisons of protein levels were performed between HRAS^WT and mutant HRAS^Q61R, both relative to EV. Error bars, s.d. of mean (n = 3). n.s. = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; two-tailed unpaired t-test. b Representative micrographs of cells cultured in three-dimensional basement membrane for 10 days showing the effects of EV, HRAS^WT, or mutant HRAS^Q61R expression in MCF-10A^P, MCF-10A^H1047R, and MCF-12A cells on growth and glandular architecture (scale bars, 400 µm). c Representative micrographs of E-cadherin, vimentin, and calponin immunohistochemical expression in a HRAS and PIK3CA mutant adenomyoepithelioma (AM32). Note the bi-layered glandular architecture where E-cadherin is preferentially expressed in the inner epithelial layer, whereas vimentin and calponin decorate the outer myoepithelial layer (scale bars, 50 µm). d, e Representative confocal images of immunofluorescence analysis of d E-cadherin (green) and vimentin (red) and 4,6-diamidino-2-phenylindole (DAPI, blue; scale bars, 25 µm), and e calponin (green) and DAPI (blue; scale bars, 50 µm) of MCF-10A^P, MCF-10A^H1047R, and MCF-12A cells stably expressing EV, HRAS^WT, or mutant HRAS^Q61R grown in three-dimensional basement membrane culture for 10 days. In b, d and e, experiments were independently performed at least three times