Overexpression of SPHK1 in HS1 cells enhances their proliferation and cloning efficiency and confers tumorigenicity. (A) Number of colonies formed by control 663-B1 and 663-B2 cells and SPHK1-transfected 663-K3 and 663-K39 cells. Five hundred cells were inoculated in semisolid medium containing 1% serum and Epo (1 U/mL) and the colonies were numbered after 8 days. The representative plotted data are shown as the average colony number (±SD) from four experiments in duplicate, determined in a doubled-blinded manner. ^* indicates statistical significance by Student t test: P < .005 compared with the control. (B) Colony-formation assay with control 663-B2 cells and MT-SPHK1-transfected 663-K3 cells. Images were captured as in Figure 4C, except for given magnifications. (C) Percentage of recipient mice bearing tumors after subcutaneous injection of control 663-B2 cells, SPHK1-transfected 663-K3 and 663-K39 cells, and 921 HS2 cells (7 mice/cell line). (D) Expression of the MT-SPHK1 and endogenous SPHK1 in the subcutaneous (sc) and spleen tumor (sp) isolated in the same nude mice grafted with 663-K39 cells was analyzed by Western blotting using anti-SPHK1 antibodies.