Figure 4.

KLF4/5 cooperate to promote malignant properties in M6 cells, a HER-2-overexpressing mammary cancer model. (a) Anchorage independence was assessed by incubation of the indicated cell populations in soft agar for 14 days (N=3, bars, S.E.). (b) Anchorage independence of HMLE cells expressing ectopic KLF4/5 was determined as previously described. Empty vector (–) served as a control so that all cell populations were treated with equal volumes of lentiviral supernatant. (c) Cells were injected into the mammary gland of female athymic mice and tumor xenograft volume was monitored over a period of several weeks (left panel, N= 5; bars, S.E.). Similar effects on tumor burden were obtained using distinct shRNAs for the suppression of each KLF (right panel, N= 5; bars, S.E.). (d) Cell death was determined by Trypan blue exclusion following 24 h of matrix deprivation for the indicated cell populations (N=3; bars, S.E.). (e) As an independent method, cell death because of matrix deprivation in M6 cells was determined by fluorescence microscopic imaging of DAPI/PI-stained cells. Results were quantitated using ImageJ (two-tailed t-test; N=3; bars, S.E.). *P<0.05; **P<0.01; ***P<0.001