Integrin-β3–mediated signaling enhanced expression of stemness-related genes by cooperating TPO presence. (A) DNA array analysis was performed using CD48⁻KSL cells after culture. The cells were sorted after culturing CD34⁻KSL cells for 5 days with 2C9.G2 or IgG in the presence of SCF and/or TPO. (B) Genes whose expression had changed from the DNA microarray data and genes that showed > 1.4-fold up-regulation (with TPO: 2231 genes or SCF + TPO: 3354 genes) or down-regulation (with TPO: 4349 genes or SCF + TPO: 2630 genes) in 2C9.G2-treated cells were selected for extraction. This was followed by extraction of genes included in both populations (up-regulation, 605 genes; down-regulation, 695 genes). In addition, to clearly focus on the effect of the combination by TPO and β3-integrin signaling, genes only showing > 1.0-fold up-regulation (362 genes) or down-regulation (336 genes) in the presence of SCF were excluded. This left 243 genes that were up-regulated and 359 that were down-regulated in HSCs by 2C9.G2 in the presence of TPO. (C) Expression of candidate genes involved in the maintenance of LTR activity of HSCs was assessed using real-time RT-PCR with 2C9.G2- or IgG-treated CD34⁻KSL cells cultured in the presence of TPO. The graphs represent mRNA expression of the indicated genes. Data are mean ± SD; n > 3. **P < .05. (D) Fresh (uncultured) CD34⁻KSL cells obtained from the BM of WT or Y747A mice were also subjected to real-time RT-PCR to examine expression of these genes. The graphs represent mRNA expression of the indicated genes. Data are mean ± SD; n > 3. **P < .05.