Figure 5From: Cdc42 overexpression induces hyperbranching in the developing mammary gland by enhancing cell migrationCdc42 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.Back to article page