Correlation of SPHK1 expression with invasiveness of esophageal carcinoma cell lines. (A, B) SPHK1 expression was analyzed by RT-PCR and immunofluorecence in the esophageal carcinoma cell lines: KYSE30, KYSE150, KYSE510, KYSE2, EC9706 and NEC. (B) In these cell lines, SPHK1 was detected by PCR after 35 cycles. (C) Invasion assays were carried out by seeding 1 × 104 cells in a 24-well transwell unit on polycarbonate filter with 8-μm pores coated with Matrigel. After a 24-h incubation period, the cells that had passed through the filter into the lower wells were stained, counted and photographed. All experiments were performed in triplicate and repeated twice. KYSE2 and KYSE30 cells showed the highest SPHK1 expression at the mRNA and protein levels, and they were also the most invasive among the six cell lines.