Fig. 7

Loss of Spy1 delays tumour onset. (a) Cells were isolated from tumours developed following mammary fat pad transplantation and mammosphere formation assays were performed. Sphere forming efficiency was quantified in primary (left panel) and secondary (right panel) mammospheres (n = 3). (b) Primary cells from Spy1, p53 null and Spy1 p53 null tumours were injected into fat pads of wildtype mice for a limiting dilution assay. (c) Primary tumour cells from Spy1, p53 null and Spy1 p53 null tumours were treated with a panel of therapeutic agents for 24 h (top panel) and 72 h (bottom panel). Trypan blue exclusion analysis was performed to assess total number of live cells (n = 3). (d) Primary cells from p53 null tumours were infected with lentivirus containing control (pLB) or Spy1 knockdown (pLB-shSpy1) and cultured as mammospheres. Sphere forming efficiency was assessed and quantified for primary and secondary spheres (n = 3). (e) p53 null primary tumour cells infected with control (pLB) or knockdown of Spy1 (pLB-Spy1) were injected into fat pad of FVBN/J mice. Timing of tumour onset is depicted (n = 10). Errors bars represent SE; Student’s T-test (a, c, d), Mann-Whitney (e). *p < 0.05, **p < 0.01, ***p < 0.001. Single asterisks over bar indicates significance to DMSO within genotype in panel c)