Figure 4

BRF2 and DSN1 amplifications are potential driver genetic alterations in HER2-negative breast cancer cells. (A) Nuclear subcellular localization of BRF2 and DSN1 in NIH3T3 (top) and MCF10A (bottom) cells expressing BRF2-ZsGreen and DSN1-ZsGreen (scale bar, 25 μm). (B) Foci formation assay of NIH3T3 cells expressing vector control, BRF2 or DSN1 protein. Cells were fixed and stained with crystal violet 21 days after plating, and the foci were quantified (see Materials and methods). *P < 0.05, unpaired t-test. Error bars represent standard deviation of mean. (C) Anchorage-independent growth of MCF10A cells expressing vector control, BRF2 or DSN1 protein. Quantification was performed using an MTT assay (left) or by defining the number and size of colonies (right). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unpaired t-test. Error bars represent standard deviation of mean. (D) Impact of empty vector, BRF2 and DSN1 expression on growth and glandular architecture of MCF10A (top) and MCF12A (bottom) cells grown in three-dimensional basement membrane cultures (scale bar, 500 μm).