Wt1 heterozygous loss of expression has a time-dependent impact on 5-hmC levels. (A) DNA 5-hmC content of splenocytes from control, Wt1^fl/+, and Wt1^fl/fl mice from young chronic, young acute, and old acute cohorts and from 1 Wt1^fl/+ aged primary mice (elderly) was established by dot blots. Data are representative of 3 experiments, with 7 animals per arm in total. Methylene blue (MB) was used as a loading control for all experiments. (B) Ratio of 5-hmC/MB spot intensities from 3 experiments. (C) DNA dot blots for 5-hmC in genomic DNA from splenocytes from Wt1^fl/+ mice in the young chronic cohort treated with increasing vitamin C (Vit-C) concentrations (0-1 µM) for 6 days. Data are representative of 3 experiments, in triplicate. (D) WT1 overexpression in Tet2-knockdown c-Kit⁺ cells delayed Tet2-associated leukemia, as reflected by the percentage of circulating GFP⁺ cells, WBC counts, and survival curves of Tet2^fl/fl cells infected with WT1-expressing (WT1 OE) viruses compared with controls (EV). Data are mean ± standard error of the mean (B-E). *P < .05, **P < .01, ****P < .0001, 2-way analysis of variance (B-D) or Mantel-Cox test (D).