Dynamic changes in S1P concentrations in the BM and PB regulate G-CSF–induced mobilization. (A-C) WT and Sphk1 deficient mice were injected with PBS (control) or G-CSF (5 consecutive subcutaneous injections and evaluation 3-5 hours after the last injection). The numbers of WBCs (A), CFU-Cs (B), and primitive SKL (C). Left, representative FACS plots of Lin− PB-MNC. The numbers in the top right quadrant of each plot represent SKL cells per 1 × 106 PB-MNC.Right, total PB-MNC cell analysis per milliliter of blood are shown. (D,F,G,J) Mice were injected with PBS (control), G-CSF (5 consecutive subcutaneous injections and evaluation 3-5 hours after the last injection), G-CSF+Rapamycin (5 consecutive subcutaneous injections of both agents) or G-CSF+Akt inhibitor (5 consecutive intraperitoneal injections together with G-CSF). MS quantification of S1P levels in the BM condition medium (BM-CM; D) is shown. (E) Quantitative Real-Time PCR analysis of Sphk1 (S1P producing enzyme) and Sgpp1 (S1P degrading enzyme) mRNA levels in PBS (control) or G-CSF (single subcutaneous injection and evaluated after 30 minutes) total BM cells are presented. Quantitative Real-Time PCR analysis of Sphk1 (F) and Sgpp1 (G) mRNA levels in total BM cells are shown. (H) Quantitative Real-Time PCR analysis of Sphk1 mRNA levels in mice treated with PBS (control) or G-CSF (single subcutaneous injection and evaluated after 30 minutes or 24 hours) in PB-MNC are presented. (I) Plasma S1P ELISA levels in PBS (control) or G-CSF (single subcutaneous injection and evaluated after 30 minutes or 4 hours and 3, 4, or 5 consecutive subcutaneous injections and evaluation 3-5 hours after the last injection) are presented. (J) Plasma S1P ELISA levels are shown. Indicated values are presented as mean ± SE in 3 independent experiments, a total of 6 mice in each group (*P < .05, **P < .01).
