Bahcc1 depletion suppressed myeloid immortalization of LT-HSCs by conditional expression of MLL-ENL. (A-B) Western blot analyses of Bahcc1 in 293 cells (293^Bahcc1) that stably expressed 3XFLAG-tagged Bahcc1 (A), and in shRNA-transduced 293^Bahcc1 cells (B). 293 cells transduced with pcDNA3.1 vector alone were used as a negative control in panel A. shRNAs against luciferase (shLuc, a negative control) and Bahcc1 (shB-2 and shB-8) were transduced into 293^Bahcc1 cells in panel B. Immunoprecipitants with anti-FLAG antibody were blotted with anti-DDDDK-tag antibody to detect expression of Bahcc1 (upper panels). α-Tubulin was analyzed in total lysates as an internal control (bottom panels). (C-D) Relative-relative expression levels of Bahcc1 (C) in immortalized LT-HSCs with shRNA transduction and clonogenicity (D) of the cells. (E-F) Representative FACS plots and quantification of CD11b⁺/Gr-1⁺, Gr-1⁻/c-kit⁺, and apoptotic (annexin V⁺/7AAD⁻) subpopulations by FACS (E), and RT-qPCR of Hoxa9, Meis1, and Evi1 (F) in colony-forming cells derived from immortalized LT-HSCs with depletion of Bahcc1. Bar graphs show the mean with SD of at least 3 independent experiments. ^∗P < .05; ^∗∗P < .01; ^∗∗∗P < .001; ^∗∗∗∗P < .0001; ns, not significant (ANOVA followed by Dunnett multiple comparisons for panels D-F).