Msi1 knockdown reduces spheroid colony formation. MCF-7 and T47D cells were transduced with either an Msi1 shRNA (Msi1 shRNA) or control shRNA (Contr shRNA) as in Figure 3A. A. Cells were seeded at varying dilutions into 96-well ultra low attachment plates in DMEM media without serum and supplemented with growth factors, and the number of colonies determined after 3 weeks. Msi1 KD significantly reduces colony formation in MCF-7 and T47D cells (***P < 0.001; **P < 0.01; *P < 0.05; two-tailed Student's t test; N = 12). B. Msi1 KD reduces spheroid colony size. Upper panel, spheroid colony morphology by fluorescence (GFP) and Brightfield microscopy; Lower panel, colony diameter at a density of 25 cells/well. Msi1 KD significantly reduced colony size (P < 0.001; two-tailed Student's t test; N = 10). C. After 10 days in spheroid culture, MCF-7 cells were replated as monolayer cultures and cell growth was measured by sulforhodamine B staining at 24, 48 and 72 hr. Msi1 KD reduces the growth of MCF-7 cells 50% vs. control cells (Contr shRNA). Each value represents the mean ± S.E. of 4 samples (**P < 0.01, *P < 0.05 by the two-tailed Student's t test.