Fig. 1.

Efficacy of 12/15-lipoxygenase (12/15-LO) cholesterol-tagged small interfering RNA (chol-siRNA) on 12/15-LO levels in vitro and distribution in vivo. A: murine mesangial cells (MMCs) were transfected with cholesterol-tagged siRNA 233 and 826 mixture or mismatched (MM) oligo mixture, and 12/15-LO gene expression was examined by RT-PCR with 18S internal control. Significant reduction (almost 60%) of 12/15-LO mRNA level was observed. Ctrol, MMC without transfection; MM233+826, mismatched oligo transfection; siRNA233+826, mixture of two 12/15-LO siRNAs. B: Western blot showing that siRNA 233 and 826 mixture inhibited 12/15-LO protein expression in MMC, while MM siRNA had no effect relative to control. C: siRNAs targeting 12/15-LO or MM mixture. Four hundred micrograms were delivered to normal DBA/2J mice. Seventy-two hours later, the mice were killed and 12/15-LO mRNA levels in various tissues were quantified by real-time quantitative PCR (QPCR) as described in materials and methods. Data show that the siRNA significantly decreased 12/15-LO gene expression in the spleen, kidney, liver, and heart, indicating broad tissue biodistribution of the siRNAs.*P < 0.05, #P < 0.01 vs. MM; n = 3. D: representative Western blots showing clear reduction of 12/15-LO protein levels in the renal cortex and liver of mice treated with 12/15-LO siRNA relative to MM.