Suppression of CD80 and CD86 expression and CHS by PU.1 siRNA. Murine BMDCs were transfected with 2 μg of PU.1 siRNA or negative control siRNA (N.CTRL; A-B). (A) After a 24- or 48-hour culture, total RNA was extracted from each transfectant, and the amounts of PU.1, CD80, CD86, and β-actin mRNAs were analyzed by ABI7500. PU.1, CD80, and CD86 mRNA levels are represented as a ratio relative to those in negative controls. Results are expressed as means + SD for 3 PCRs performed in duplicate. (B) After a 48-hour culture, cells were harvested and subjected to analysis for CD80 or CD86 by flow cytometry. Solid line histogram represents cells with each antibody. Dotted line histogram represents negative control with 2.4G2 alone. Representative results of 3 independent experiments are shown. CHS against TNCB was induced as described in “Introduction of contact hypersensitivity and treatment with siRNA” (C-D). Nonsilencing control (N.CTRL), PU.1 siRNA, CD80 siRNA, CD86 siRNA, or a CD80/CD86 siRNA combination mixed with a cream-based ointment was applied (C), or each siRNA was subcutaneously injected (D) and the ear was challenged with TNCB. Ear swelling was measured at 24 and 48 hours after challenge. Sterile water mixed with the cream-based ointment was used as a control (vehicle, C). Diethylpyrocarbonate-treated water was subcutaneously injected as a control (vehicle, D). Data are representative of 2 or 3 independent experiments (n = 4∼6) and significant differences from the nonsilencing control siRNA group (*P < .1; **P < .01) were observed. Forty-eight hours after the challenge, ear swelling in the PU.1 siRNA group was significantly reduced compared with the CD80 siRNA group (*P < .05).