Skip to main content
. 2013 Sep 3;123(10):4144–4157. doi: 10.1172/JCI68951

Figure 2. FTY720 and its nonimmunosuppressive derivatives decrease survival and self-renewal of quiescent CML HSCs.

Figure 2

(A) CFC/replating results showing replating efficiency of single CFC colonies from CD34+CD38– CML and NBM cells ectopically expressing HA-tagged PP2Ac or treated with FTY720 or imatinib. LTC-IC results show number of colonies derived from clonogenic assays of CML and NBM cells cultured for 6 weeks and exposed to the indicated drug during the first week of culture. (B) Number of quiescent CFSEmaxCD34+ cells (gated cells; inset) in untreated and drug-treated CFSE-labeled CD34+ NBM and CML BM cells. Annexin-V/7-AAD staining shows percent CFSEmaxCD34+ or CD34+CD38– CML and NBM cells undergoing apoptosis. (C) Intracellular levels of nonphosphorylated and phosphorylated FTY720 in 32D-BCR-ABL cells treated with FTY720 (2.5 μM) or FTY-P (2.5 μM), as measured by LC/ESI/MS/MS. (D) Left: PP2A phosphatase activity assay (mean ± SD) performed on 32D-BCR-ABL cells treated with FTY720 or with its nonimmunosuppressive derivatives (S)-FTY720-OMe, (S)-FTY720-regioisomer, and OSU-2C. Right: Confocal microphotographs demonstrating lack of phosphorylation of the FTY720 derivatives by showing the distribution of GFP-tagged S1PR1 after treatment with FTY-P (positive control), (S)-FTY720-OMe, (S)-FTY720-regioisomer, and OSU-2S. Original magnification, ×630. (E) Percent CD19+B220+ B cells in the PB of FVB/N WT mice after i.p. injection with FTY720, (S)-FTY720-OMe, (S)-FTY720-regioisomer, or OSU-2S. Molecular structures of FTY720, (S)-FTY720-OMe (48), (S)-FTY720-regioisomer (48), and OSU-2S (49) are also shown. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test.