Overexpression of SPHK1 protects HS1 cells from apoptosis. (A) Proliferation of pEF-BOS-transfected cells (663-B2) and MT-SPHK1-transfected cells (663-K3 cells) in a culture medium containing 1% serum and Epo (1 U/mL) in the presence or absence of DMS 10 μM. The graphs are means ± SD of 5 independent experiments performed in duplicate. ⋄ indicates 663-B2 + 1% FCS + Epo; ♦, 663-B2 + 1% FCS + Epo + DMS; □, 663-K3 + 1% FCS + Epo; and ▪, 663-K3 +1% FCS + Epo + DMS. (B) Percentages of dead cells were determined by trypan blue exclusion assay on 663-B2 and 663-K3 cells cultured for 24 hours and 48 hours in a medium containing 1% serum and Epo (1 U/mL) in the presence or absence of 10 μM DMS. Data are means ± SD of 5 experiments in duplicate. ^* indicates statistical significance by Student t test: P < .05 compared with the control. ▦ indicates 663-B2; □, 663-B2 + DMS; ▪, 663-K3; and ▨, 663-K3 + DMS. (C) Detection of fragmented and condensed nuclei in apoptotic cells by fluorescence microscopy. Representative images after Hoechst staining of 663-B2 and 663-K3 cells cultured in a medium containing either 10% or 1% serum and Epo (1 U/mL) for 48 hours. Magnification, × 63. Cells were observed using a Nikon Eclipse TE300 microscope (Nikon, Champigny sur Marne, France) with 40 × objective magnification. Images were acquired with Nikon Coolpix 950 and processed using Adobe Photoshop (Adobe Systems, San Jose, CA). Three different fields (500 cells per field) were scored. (D) Processing of caspase-3 and activation of AKT and ERK1/2 in 663-B2, 663-K3, and 663-K39 cells cultured in a medium containing 1% serum and Epo (1 U/mL) in the presence (+) or absence (-) of DMS (10 μM) for 24 hours. Cell lysates were subjected to immunoblotting with antibodies indicated on the left of the blots. Blots were reprobed with a β-actin antibody to control loading of the gels. Western blots are from a representative experiment. Similar results were obtained in 3 independent experiments.