p53 knockdown by siRNA. (a) Western blot analysis. Cultured fibroblast-like synoviocytes (FLS) were transfected with 1, 2.5, or 5 μg of siRNA or non-silence scrambled siRNA (sc) as described in the Materials and methods section. Mock-transfected cells were treated in the same manner except that no siRNA was added. FLS were then incubated for 3 or 5 days and Western blot analysis was performed. The p53 level was decreased on day 3 and day 5, especially with the higher amounts of siRNA. For subsequent experiments, 2.5 μg of siRNA for 3 days was chosen as an optimum condition. Two FLS lines were studied with similar results. (b) Immunohistochemistry staining of p53 protein expression in rheumatoid arthritis FLS. Transfected FLS were seeded into four-well chamber slides, cultured for 5 days, and evaluated by immunohistochemistry. Prominent staining was observed in the positive control (colon tumor cell line HCT116) as well as scrambled siRNA-transfected FLS. The number of p53-positive cells was significantly reduced after transfection with 1 μg (not shown) or 5 μg of p53 siRNA (p < 0.0001 for p53 siRNA-transfected cells compared with scrambled siRNA). Percentages of p53-positive cells are shown as means ± SEM. Two FLS lines were studied with similar results. An irrelevant control antibody was positive in less than 1% of cells (not shown).