Ex vivo inhibitor treatments implicate RhoA/ROCK pathway in NMII activation during branching. (A) Explants derived from K14-Cre;R26R-mTmG embryos were cultured for 5 d and treated 24 h with 20 µM blebbistatin (right) or vehicle (left) and after whole-mount staining of K14 (cyan, upper pair of glands) or K8 (cyan, lower pair of glands) visualized together with epithelial mGFP (magenta) by confocal imaging. (B and E) To assess the roles of ROCKI/II and MLCK, ex vivo cultured mammary glands with epithelial mGFP expression (K14-Cre; R26R-mTmG), were also imaged before and after treatment with 50 µM Y27632 (B) or 20 µM ML-7 (E) together with vehicle (mGFP = gray). (C, D, F, and G) The number of new tips was quantified (C and F), and the fold increase in growth area measured (D and G) from wide-field images of the mGFP-labeled epithelium before and after treatment (n^{control for Y27632} = 27 glands from 13 explants, n^Y27632 = 29 glands from 13 explants, four experiments; n^{control for ML-7} = 31 glands from 15 explants, n^ML-7 = 37 glands from 15 explants, four experiments). (H) To assess the effects of blebbistatin treatment on cell proliferation, control and treated mesenchyme-free sprouting organoids were labeled with EdU for 1 h at the end of the 24-h culture period, fixed for staining and confocal imaging (maximum intensity projections of EdU in white and Hoechst in blue). The percentage of EdU+ cells is indicated on the top left corner (n^control = 3 rudiments, n^treated = 2 rudiments, one experiment. Data shown represents the median (line) with 25th and 75th percentiles (hinges) plus 1.5× interquartile ranges (whiskers). Statistical significance was assessed with the Wilcoxon’s rank sum test; ****, P ≤ 0.0001.