Figure 6.
Mutation of the Smad3 linker phosphorylation sites in 4T1 cells reduces putative stem cell population and tumor initiating frequency of cells from the initial xenograft in limiting dilution. A, schematic representation of the initial and secondary xenograft. 4T1 cells (5 × 105) infected with Ad-Smad3, Ad-3SA, or Ad-EPSM were injected orthotopically into Balb/c mice (5 mice per group). Parental cells were used as controls. Three weeks after injection, single cells from the dissociated tumors were subjected to FACS analysis using the Aldefluor assay (B), and Aldefluor-positive cell population (%) was determined (C). Each bar represents the mean ± SD from four samples per group. **, P< 0.01, compared with Ad-Smad3–infected cells. D, tumor-initiating frequency of secondary tumors formed by injection of cells from the primary tumors. These experiments were conducted three times with similar results.