Fig. 3

Spy1 overrides p53 to promote MaSC expansion. (a) Primary mammary epithelial cells were isolated from inguinal mammary glands of MMTV-Spy1 and littermate control mice and cultured as mammospheres in the presence and absence of 2.5µM Nutlin3. Sphere forming efficiency of primary mammospheres (left panel) and secondary mammospheres (right panel) is depicted (n = 3). (b) Control (pEIZ) or Spy1 overexpression (Spy1) MCF7 cells were treated with 2.5µM Nutlin3 and cultured as mammospheres. Sphere forming efficiency in the presence or absence of Nutlin3 is depicted. (c) siRNA mediated knockdown of p53 was performed in primary mammary epithelial cells from control and MMTV-Spy1 inguinal mammary glands, and cells were cultured as mammospheres. Sphere forming efficiency is depicted (n = 5). (d) MCF7 cells were lentivirally infected with pEIZ, pEIZ Spy1 (Spy1), pLKO, pLKO shSpy1 (shSpy1.1, shSpy1.2), p53 or shp53. MCF7 cells with Spy1 overexpression (left panel) or Spy1 knockdown (right panels) in the presence or absence of p53, were cultured as mammospheres. Sphere forming efficiency was assessed and quantified (lower panels). Representative images shown in upper panels. Scale bar = 200 μm; n = 3; Errors bars represent SE; Student’s T-test. *p < 0.05, **p < 0.01, ***p < 0.001