Figure 3. Effect of CerS2 siRNA on ceramide synthesis.

SMS-KCNR neuroblastoma cells were treated with 20 nM CerS2-specific and control siRNA oligonucleotides. At 72 h after siRNA treatment, cells were lysed, and microsomes or RNA were isolated from the lysates. (A) RT-PCR results with CerS1-, CerS4-, CerS5- and CerS6-specific primers. The error bars represent the range for two independent experiments. (B) Western blot analysis on microsomes with a CerS6-specific antibody. Na⁺/K⁺-ATPase was used as a loading control. The Western blots are representative of four independent experiments. (C) Microsomes were used for in vitro ceramide synthase assay with C₁₆ fatty acyl-CoA and C₁₇ sphingosine substrates. The error bars represent the range for two independent experiments. (D) At 48 h after the siRNA treatment, cells were labelled with 1 μM C₁₇ sphingosine for 30 min. Where appropriate, 30 μM FB1 was added to the samples 30 min before cells were labelled with C₁₇ sphingosine. Lipids were extracted from the cell pellets and subjected to MS analysis. Sphingolipids were normalized to cellular lipid phosphate. The error bars represent the range for two independent experiments.