Figure 3.

Generation of sphingosine kinase knockdown cell lines. (A) mRNA levels of SK1 and SK2 in Jeko and SP53 MCL lines were normalized to 18S expression. (B) SP53 cells were stably transfected with shRNA plasmids to knock down expression of sphingosine kinase 1 (SK1) or with a scrambled shRNA control plasmid. Two clones were selected and used for experiments (SK1-KD1 or SK1-KD2). Knockdown of SK was confirmed by quantitative real-time PCR (qPCR). SK1 and SK2 levels were assessed relative to 18S. Expression in the parental cell line (SP53) was compared to the scrambled shRNA control and the SK1-KD clones. (C) Reduction of SK results in decreased S1P production. S1P levels in cell lysates were measured by ELISA. (D) Control (SP53), SK-KD1, and SK-KD2 cell growth was monitored by WST-1 using absorbance at 440 nm. Error bars are contained within the points. Data was analyzed by a two-tailed t-test. * p < 0.05; ** p < 0.001.