Hif-1α deletion accelerates the AE9a malignant phenotype in secondary recipients. (A) Kaplan-Meier survival curve of secondary recipients from 2 independent experiments transplanted with cells derived from leukemic mice of both genotypes (Hif-1α^Δ/Δ, n = 7; Hif-1α^fl/fl, n = 9). The log-rank (Mantel-Cox) test was used to assess statistical significance. The development of the disease was measured by the percentage of transduced cells (GFP⁺) (B), myeloid cells (Gr1⁺ and/or Mac1⁺) (C), and Lin^– cells (D) in PB 3, 7, and 10 weeks after transplantation (Hif-1α^Δ/Δ, n = 11; Hif-1α^fl/fl, n = 18). Eleven weeks after transplantation, mice were sacrificed and disease analyzed by measuring several parameters: total WBC (E), spleen and liver weight (F), and GFP⁺ and Lin^– cells in the BM and spleen (G) (Hif-1α^Δ/Δ, n = 7; Hif-1α^fl/fl, n = 9). A 2-way ANOVA test was used to assess statistical significance along the different time points. (H) Representative FACS plots of spleen cells from diseased mice showing that the GFP⁺ cells are all included in the Lin^– malignant population. The differentiated populations are stained with the following antibodies: CD3 for T cells (T), B220 for B cells (B), and Gr1/Mac1 for myeloid cells (M). Plots represent mean ± SEM. Unless otherwise stated, 2-tailed Student t test was used to assess statistical significance. *P < .05, **P < .01, ***P < .001.