Figure 4

Runx1 haploinsufficiency alters expression of key genes modulating cytokine response and hematopoietic stem/progenitor cells mobilization. (a) Expression profiles of Pias-, Socs-family genes and Ptprc within the KSL cells from the BM. Expression of HPRT1 was used as endogenous control. (n=3/genotype). (b) Expression of Pias3 and Socs3 within the KSL cells from the spleen. Expression of HPRT1 was used as endogenous control (n=5/genotype). (c) Occupancy of RUNX1 at PIAS3 gene locus in human CD34+ cell-derived megakaryocytes. Black arrowheads represent RUNX1-binding sites. (d and e) Luciferase activity in HL-60 cells after co-transfection of either PIAS3 (d) or CXCR4 (e) reporter with the indicated RUNX1, RUNX1^R174Q (R174Q), RUNX1^Y260X(Y260X) and CBFB plasmids. Black arrowheads on the luciferase constructs indicate the position of RUNX1-binding sites. Luciferase activity was normalized to Renilla internal control. Three independent experiments were performed. (f) Relative expression of Cxcr4 mRNA in KSL cells. Mice were treated with 250 μg/kg/day G-CSF in vivo stimulation for three consecutive days. All data represent mean±s.d. (Runx1^+/+mice phosphate-buffered saline (PBS) control, n=2; Runx1^+/− mice PBS control, n= 2; Runx1^+/+mice G-CSF-treated, n= 3; Runx1^+/+mice G-CSF-treated, n= 3). Asterisks represent significant differences (*P<0.05, **P<0.01 and two-tailed Student’s t-test).