Fig. 7
The effects of HOXA9 overexpression from D4 on hematopoiesis are closely related to NF-κB signaling and might be caused by a change in the cell cycle state. a qRT-PCR detection at D14 revealed that expression levels of NFKB1 and NFKB2 were significantly enhanced in D4-induced HOXA9/hESCs co-cultured with AGM-S3 cells, which indicated that at the late stage NF-κB signaling was up-regulated by HOXA9 overexpression. b D4-induced HOXA9/hESCs co-cultured with AGM-S3 cells were treated with 10 nM QNZ (an NF-κB signaling pathway inhibitor) or 20 nM siRNA against NFKB1, and flow cytometry analysis at D14 revealed that QNZ or siRNA against NFKB1 attenuated the positive effects of D4-induced HOXA9/hESCs on CD34 − CD43+, CD34 + CD43+, CD34 − CD45+, CD34 + CD45+ populations and the inhibitory effects on CD71 + GPA+ population. c Analyses of cell cycle state revealed that HOXA9 induction from D4 increased the proportion of cells in S phase in co-cultured cells or CD45+ cells expressing HOXA9 (the GFP+ fraction); this effect was eliminated by treatment with QNZ or siRNA against NFKB1. The experiments were repeated three times, and P < 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)