Induced Sirt1 deletion promotes hematopoietic progenitor cell expansion and deregulation of Hoxa9 in vitro. (A) BM culture of Sirt1-E4^fl/fl, ERT2-Cre (SIRT1^Δ/Δ) and Sirt1-E4^fl/fl (WT) cells in indicated growth factors after 4-OHT–induced Sirt1 ablation. Cell numbers were counted in duplicate at indicated passage time points, and similar results were obtained in three independent experiments. (B) Fraction of BrdU⁺, c-Kit⁺, Sca-1⁺ SIRT1^Δ/Δ, and WT cells cultured in the indicated growth conditions. BrdU incorporation was measured after 5 d of culture after 6 h of BrdU administration. The fraction of BrdU⁺ cells was normalized to WT for each culture condition, and samples were analyzed in triplicate. (C) Gene expression array analysis of FACS-sorted c-Kit⁺Sca-1⁺ERT2-Cre (Cre) control and SIRT1^Δ/Δ cells cultured in the presence of SCF, TPO, FLT3L, and IL-6. Relative expression of SIRT1^Δ/Δ compared with Cre is shown for probe sets representing definitive hematopoiesis genes. Dashed lines depict twofold expression changes (up or down). (D) Hox gene expression analysis of microarray samples from C. (E) Analysis of expression levels of Hoxa9, Hoxa10, Hoxa7, and Sirt1 exon 4 in unsorted WT and SIRT1^Δ/Δ cells from an independent 4F culture. RNA was isolated 5 d after Sirt1 deletion and subjected to qRT-PCR, and samples were analyzed in triplicate and normalized to β-actin and Rpl13a. (F and G) Analysis of expression levels of Hoxa9, Hoxa10, and Sirt1 exon 4 in WT and SIRT1^Δ/Δ cells cultured in the presence of SCF (F) or SCF and FLT3L (G). RNA was isolated 5 d after Sirt1 deletion, and samples were analyzed by qRT-PCR in triplicate and normalized to Gapdh and β-actin. Error bars represent SD. *, P ≤ 0.05; **, P ≤ 0.01.