Inhibition of FOG-1 expression by siRNA resulted in upregulation of β-chain promoter activity. (A) Quantification of FOG-1 mRNA in siRNA transfectants using real-time RT-PCR. Ba/F3 cells (2 × 106) were transfected with 5 μL of 20 μM FOG-1 siRNA or shuffled control siRNA. After 20-hour culture, total RNA was extracted from each transfectant, and the amount of FOG-1 and GAPDH mRNAs was analyzed by ABI7500. FOG-1 mRNA levels are represented as the ratio to that of control (without siRNA). (B) Western blot analysis of transfectants. After 44 hours of culture, lysates (5 × 105 cells per lane) were analyzed using anti-FOG-1 or anti-YY1 Abs. (C) Transcription activity of the β-chain promoter. One microgram of reporter plasmid was introduced into Ba/F3 cells with or without 2.5 or 5 μL of 20 μM siRNA or shuffled control siRNA. The ratio of luciferase activity of each transfectant to that of control transfectant (without siRNA) was represented as fold activation.
