Figure 3.

Transfection of murine macrophage RAW 264.7 cells. Three siRNA candidates against murine COX-2 were evaluated; siCOX2-1, siCOX2-2, and siCOX2-3. siRNAs were mixed with Trans-IT-TKO^® and added at 50nM final siRNA concentration to cell cultures and incubated for 24 hours. Afterwards, transfected cells were stimulated by LPS (100 ng/mL) for 6 hours to induce COX-2. (A) RNA was extracted from cells followed by cDNA synthesis, which served as template for QPCR. COX-2 mRNA levels was investigated by QPCR. (B) Protein was extracted from cells and western blot analysis was performed to detect COX-2 protein levels. ß-actin was run in parallel to ensure equal protein loading. (C) PGE₂ levels in cell culture media evaluated by ELISA. Results are means ± SEM. WT: wildtype, LPS: lipopolysaccharide, siEGFP: negative coding control siRNA, NC: non-coding control siRNA, siCOX2: siRNA targeting murine COX-2.