Figure 2. GSK3β is preferentially involved in HSPC motility.

(A) BM MNCs were pretreated with 1 μM BIO-A (a selective GSK3β inhibitor) or equivalent DMSO for 1 hour and then loaded into Transwells. Migration was assessed for 2 hours as being either spontaneous or toward 125 ng/ml CXCL12 (n = 8). (B) CXCL12-induced migrating BM MNCs were washed and then seeded into methylcellulose colony assay to measure CFU cells, indicating HSPCs. (C) In addition, LK cells were measured among migrating BM MNCs (n = 3–5) (C), and CD34^–LSK cells were measured among migrating Lin^– cells (n = 5–6). (D) Following treatment with 1 μM BIO-A or equivalent DMSO for 1 hour, CXCR4 expression (fold change) was determined by flow cytometry in total BM and LK cells (n = 3). (E–G) Mice were treated with 0.6 mg/kg BIO-A or equivalent DMSO. After 1 hour, they were sacrificed and PB was obtained to measure circulating WBCs (E), as well as circulating CFU cells (F) and LSK cells (G) (n = 4). (H) LTR assay by PB HSCs. Donor mice were treated with 0.6 mg/kg BIO-A or equivalent DMSO, and after 1 hour the PB was collected. Congenic recipient mice were lethally irradiated (10 Gy) 24 hours prior to transplantation. Each recipient mouse was transplanted with 500 μl of donor PB along with 200 × 10³ host-type competitive BM cells. Donor chimerism in the recipient BM was evaluated 4 months after transplantation (n = 5–6). *P < 0.05 and **P < 0.01 in comparison with control. NS, nonsignificant.