Cdc42 overexpression enhances MEC migration and contractility and development of dysmorphic, invasive mammary acini. (A) Transwell migration assay analysis of primary MECs from line 4 and control mammary glands treated for 1 week with dox prior to isolation. Fold change (± SEM) of total cells migrated was quantified per experiment. Data represent average of four independent experiments (n = 7 control animals; n = 11 Cdc42 OE mice, *P = 0.005). (B) Western blot analysis of phosphorylated MLC on lysates prepared from whole mammary glands after 1 week of dox treatment (n = 5 animals pooled per group). Densitometry was normalized to actin loading control and is represented as fold increase compared to control. (C) Graph depicts average area of collagen gel contraction (± SEM) at 24 and 48 h post release with and without the ROCK inhibitor Y27632, (*P = 0.03). Data are representative of four independent experiments. (D) Three-dimensional culture morphogenesis assay of primary MECs from 1-week dox-treated line 4 and control mammary glands. Representative confocal images depict control and abnormal Cdc42-overexpressing acini. Arrows indicate invasive protrusions. Scale bar = 25 μm. (E) Graph depicts average fold change (± SEM) of total invasive acini per well from three independent experiments (*P = 0.007). (F) Graph shows the average fold change in dysmorphic acini per well from three independent experiments (*P = 0.008). (G) Analysis of mitotic spindle orientation in line 4 and control mammary acini (n = 50 spindles per genotype analyzed in two independent experiments). Cdc42, cell division cycle 42; MEC, mammary epithelial cell; MLC, myosin light chain; OE, overexpressing.