Figure 2
siRNAs targeted to S1PR1, R2, and R3 specifically knockdown receptor expression. (A) Combined short-interfering RNAs (siRNAs) targeted to S1PR1/R2/R3 (100 nM each) significantly reduced the mRNA expression of S1PR1 by 61.6% ± 2.9% compared to the untreated control (Cont). The levels of S1PR1 mRNA were not affected by treatment with metafectene (Meta), 200 nM of the negative control siRNA (NC), 200 nM siRNA targeted to S1PR2 (R2), or 200 nM siRNA targeted to S1PR3 (R3). The different treatment groups were normalized to their respective untreated controls. (B) Combined siRNAs targeted to S1PR1/R2/R3 (100 nM each) and siRNA targeted to S1PR2 (200 nM) alone significantly reduced the expression of S1PR2 mRNA by 74.3% ± 3.2% and 68.5% ± 2.6%, respectively. (C) Combined siRNAs targeted to S1PR1/R2/R3 (100 nM each) and siRNA targeted to S1PR3 (200 nM) alone significantly reduced the expression of S1PR3 mRNA by 76.3% ± 3.5% and 72.1% ± 1.5%, respectively. (D) The combined siRNAs targeted to S1PR1/R2/R3 did not significantly affect the mRNA levels of either S1PR4 or R5 (P = 0.88 and 0.20, respectively, ANOVA). (A-D) Values were obtained from neurons isolated from four different tissue harvests; a copy number analysis (see Kays et al. [14]) was used to quantify the values of receptor mRNA relative to the reference gene, Arbp, for the different treatments; a Kruskal-Wallis ANOVA with a Tukey post hoc test was used to determine statistical differences between the different groups where the asterisks indicate P < 0.05.