Setd2 heterozygous loss leads to decreased leukemia latency and cytarabine resistance in vivo. (A) Schema for conditional knockout of the third exon of Setd2. (B) PCR of genomic DNA of bone marrow from mice with wild-type, homozygous, or heterozygous exon 3 of Setd2 with the flanking loxP site crossed with Mx1-cre, with or without the induction of excision by polyinosinic:polycytidylic acid, as indicated. (C) Lin-Kit⁺Sca1⁺ bone marrow cells from Setd2^fl/fl, Setd2^fl/+, or Setd2^+/+ Mx1-cre mice were transduced with a MSCV-IRES-MLL-AF9-GFP construct and 200 000 GFP⁺ preleukemic cells were injected into lethally irradiated C57BL/6 recipients. Mice were monitored daily and killed when they appeared moribund or showed signs of sickness. (D) Western blot for H3K36me3 in secondary MLL-AF9 Setd2^+/+, Setd2^fl/+, or Setd2^fl/fl Mx1-cre leukemia. H3K36me3 levels were quantified and normalized to the level of total H3 in each lane. (E) A total of 200 000 bone marrow cells from mice with primary MLL-AF9 Setd2^fl/+ Mx1-cre or Setd2^+/+ Mx1-cre leukemia were injected into secondary C57BL/6 recipients. Mice with secondary MLL-AF9 Setd2^+/+ Mx1-cre (n = 9) or Setd2fl^/+ Mx1-cre (n = 24) leukemia received a single dose of cytarabine when their PB GFP was ∼30% and were rebled 12 to 16 hours later to determine the reduction in cell number, expressed as a percentage of pretreatment GFP or (F) stained with AnnexinV to determine the amount of apoptosis after treatment. (G) Mice with secondary MLL-AF9 Setd2^+/+ Mx1-cre leukemia initiated treatment with 5 days of cytarabine (n = 5) or vehicle (n = 6) when PB GFP was ∼5%. (H) Mice with secondary MLL-AF9 Setd2^fl/+ Mx1-cre leukemia initiated treatment with 5 days of cytarabine (n = 5) or vehicle (n = 6) when PB GFP was ∼5%.