Figure 6: Global loss of DNA methylation in DKO cells results in elimination of CSC population and complete loss of tumorigenicity both in vitro and in vivo. (A) DKO cells retain substantial proliferation ability in vitro. DKO, DNMT1 and DNMT3b double knockout HCT116 cells; WT, wild type cells. (B) DKO cells failed to form typical tumorspheres. (C) DKO cells completely lost colony formation ability in soft-agar plates. (D) Loss of tumorigenicity for DKO cells. 1x 102, 1 x 103 and 1 x 104 wild type HCT116 cells or 5 x 105 and 1 x 106 isogenic DKO cells lacking DNMT1 and DNMT3b were injected into both flanks of NOD/ SCID mice for assessment of tumor growth. Tumor sizes were monitored and measured weekly. N = 4.