Figure 3.

Ceramide Increases PS-ASO Activity

(A) A431 cells were pretreated with different concentrations of ceramide for 6 hr, followed by incubation with PS-ASOs targeting Drosha or Malat1 for 16 hr without the removal of ceramide. The levels of Drosha and Malat1 RNAs were determined by qRT-PCR. Percent expression relative to non-PS-ASO treated control is plotted. The error bars represent SDs from three independent experiments. p < 0.01 for 5 μM versus 0 μM (blue); p < 0.01 for 10 μM versus 0 μM (red). p values were computed by two-way ANOVA using Prism. (B) A431 cells were pretreated with 10 μM ceramide for 6 hr. Intracellular fluorescence of Cy3-PS-ASO (IONIS ID 446654) was quantified by flow cytometry at 2 hr. RFU, indicative of uptake, is plotted versus PS-ASO concentration. (C) A431 cells were treated with PS-ASOs targeting Drosha or Malat1 for 4 hr, and medium was replaced with medium without PS-ASOs but containing ceramide. After 20 hr, the levels of Drosha and Malat1 RNAs were determined by qRT-PCR. Percent expression relative to non-PS-ASO-treated control is plotted. The error bars represent SDs from three independent experiments. p < 0.01 for 10 μM versus 0 μM (red). p values were computed by two-way ANOVA using Prism.