Role of Id1 vs Id3 in leukemia development. (A) AML1-ETO and p300 colocalize at the promoter regions of Id3, but not Id2 and Id4, in chromatin immunoprecipitation sequencing analysis of Kasumi-1 cells. (B) The expression of Id1 and Id3 were examined in MIGR1, AML1-ETO, or AML1-ETO∆NHR1 (that lacks the nervy homology region 1 [NHR1] domain) transduced human CD34⁺ cord blood cells by microarray analysis (***P < .01). (C and D) In vitro doxycycline treatment decreased the expression of Id3 at both mRNA and protein levels in AE9a cells transduced with one of several inducible shRNAs against Id3. (E) The expression level of Id3 was downregulated in sorted RFP⁺GFP⁺ cells isolated from the bone marrow of the recipient mice transplanted with AE9aId1fl/flCreER cells following doxycycline or dox/tamox treatment (left panel). The expression of Id1 was downregulated in the sorted RFP⁺GFP⁺ cells isolated from the bone marrow of the recipient mice transplanted with AE9aId1fl/flCreER cells in the tamoxifen-treated groups (right panel). (F) Inducible KD of Id3 by doxycycline does not significantly affect the in vitro proliferation of AE9aId1fl/flCreER cells, with or without 4-OHT treatment (*P < .05, **P < .01). (G) Inducible KD of Id3 by doxycycline does not significantly affect the survival time of recipient mice transplanted with AE9aId1fl/flCreER cells, compared with the control shRNA (n = 10, ***P < .01).