Figure 4

Asap1negatively regulates mammary stem and progenitor cells. (A) Quantitative RT-PCR detection of Asap1 transcript abundance in MaSC/basal cells following transduction with retroviruses expressing shControl or shRNAs targeting shAsap1. Data are shown as mean ± S.E.M. (n = 3). Prox1 expression is normalized to Ywhaz1 expression relative to shControl. (B) Left panel: transduced cells were plated on an i3T3 feeder layer and cultured for 6 days to allow the formation of colonies. Right panel: histogram showing the number of colonies derived from shControl- and shAsap1-transduced cells. Data are shown as mean ± S.D for 3 independent experiments. (C) Representative whole-mount images of GFP⁺ outgrowths derived from transplantation of shControl- or shAsap1-retrovirally transduced MaSC/basal cells. Scale bar, 2 mm. (D) Morphological analysis of outgrowths. Haematoxylin and eosin staining and immunohistochemical staining for P63 and K8/K18 of outgrowths following transplantation. Scale bars, 50 μm. (E) Table of limiting dilution analysis of transplantation frequencies of MaSC/basal cells transduced with shControl or shAsap1 retroviruses. The number of transplants and resulting outgrowths is shown as well as the extent of fat pad filling by individual outgrowths.