Figure 2

Forced expression of ERAS transforms MCF10A cells. Quantification of (a) mRNA and (b) protein expression of ERAS in transfected MCF10A cells. Relative values in 2a represent the mean ± SD of three different experiments and were normalized with TBP expression. (c) Western blot showing different mobility for ERAS and other Ras proteins in several human mammary cell lines. The membrane was successively hybridized with the N-20 antibody specific for ERAS (upper band) and the 18/A panRas antibody (lower band). (d) Analysis by immunofluorescence showing the localization of ERAS protein in ERAS-transfected cells (lower row). Untransfected cells are shown in the upper row. Note that the signals for HA-tag (red) and ERAS (N20, green) antibodies overlap. DAPI staining is shown in blue. Bar, 50 μm. (e) Morphological changes of MCF10A expressing ERAS with respect to control cells; MCF10A-ERAS cells present numerous prolongations and loss of adhesion to substrate and between cells. Bar, 200 μm. (f) Disruption of normal formation of mammary acini in three-dimensional culture of ERAS-expressing MCF10A cells. The loss of the spherical and hollow morphology is shown through a series of z-stack confocal microscopy images. HA-tag antibody was used to show expression of ERAS, and cleaved caspase-3, E-cadherin, and collagen-IV antibodies were used to visualize apoptosis into acini, junction between cells, and basement membrane, respectively. All experiments were performed in duplicate, typical results are shown. Uncropped blots for b and c are presented in Supplementary Fig. S6.