Fig. 4

KLF4 re-expressing cells are outcompeted in competitive in vivo assays, especially after treatment. a Experimental procedure: NSG mice were injected with 30,000 cells from the control mixture (purple; n = 10) or wtKLF4 mixture (orange, n = 10). The control mixture consisted of a 1:1 ratio of mock- and mutKLF4-expressing cells; the wtKLF4 mixture consisted of a 1:1 mixture of mock- and wtKLF4-expressing cells. After homing was completed and the tumors were established, Dox was added to the drinking water from day 18 onward, and leukemia growth was monitored by in vivo bioluminescence imaging. Two mice from each group were sacrificed every week, and the proportions of the two subfractions in bone marrow were analyzed by flow cytometry. b Left panel: Leukemia growth as determined by in vivo imaging in mice bearing the wtKLF4 or control mixture. Right panel: Subfraction analysis by flow cytometry by gating on the subpopulation-specific fluorochrome markers T-Sapphire for the mock subpopulation and iRFP720 for either the mutKLF4 or wtKLF4 subpopulation (see Figure S2B for information about the constructs). Quantification is depicted as mean ± SEM. p < 0.01 according to a two-tailed unpaired t test. c Experiments were set up as described in A, except that Dox was administered after treatment, during tumor regrowth. When a high tumor burden was reached (day 31), mice were treated intravenously with high-dose combination chemotherapy (0.25 mg/kg vincristine + 100 mg/kg cyclophosphamide once per week, given on Mondays and Thursdays, respectively, light red background) to reduce the tumor burden to achieve minimal residual disease (MRD); at MRD (day 64), chemotherapy was stopped, and Dox was added to the drinking water to induce transgene expression. d Tumor regrowth after treatment was monitored and analyzed as described in (b)