Characterization of HS1 cells overexpressing SPHK1. (A) The pEF-BOS empty vector and expression construct for MT-SPHK1 were transfected into 663 cells or Ba/F3 cells. Expression of MT-SPHK1 in whole-cell lysates from pools of cells transfected with pEF-BOS and pMSCV-Neo (663-B2 and Ba/F3-neo), from pools of cells transfected with pEF-BOS-MT-SPHK1 and pMSCV-Neo (663-K3 and Ba/F3-SK1), and from cell clones transfected with pEF-BOS-MT-SPHK1 and pMSCV-Neo (663-K38 and 663-K39) was analyzed by Western blotting using anti-SPHK1 antibodies. Reprobing the same membrane with anti-α adaptin was used as loading control. The 49 and 54 kDa indicate the apparent molecular weights (MW) of endogenous SPHK1. (B) Western blot analysis of the expression of endogenous and/or exogenous SPHK1 in membrane and cytosolic compartments from 663 HS1, 921 HS2, 663-B2, and 663-K3 cells using anti-SPHK1 antibodies. (C) Sphingosine kinase activity in 663-B2 and Ba/F3-neo control cells and in 663-K3, 663-K38, 663-K39, and Ba/F3-SK1 cells. Data are means ± SD of duplicated samples in 3 independent experiments. (D) Overexpression of SPHK1 does not confer Epo independence to HS1 cells. Proliferation of 663 cells stably expressing MT-SPHK1 (663-K3) in culture medium containing 10% serum in the presence or absence of Epo (1 U/mL). Cells transfected with pEF-BOS vector (663-B2) were used as control. ○ indicates 663-B2 + 10% FCS + Epo; ▵, 663-K3 + 10% FCS + Epo; •, 663-B2 + 10% FCS, no Epo; and ▴, 663-K3 + 10% FCS, no Epo. Data are means ± SD of 4 experiments in duplicate.