Figure 3.

5'PPP-RNA induced RIG-I and IFNβ expression in A549 cells. (A) A549 (1 × 10⁶ cells/well) in a 6-well tissue culture plate were mock-transfected (control) or transfected with 2 μg of 5'PPP-RNA, CIAP-RNA or chemically synthesized OH-RNA using lipofectamine 2000. After 24 hr of treatment, (i) IFNβ and (ii) RIG-I mRNA expression was analyzed by real-time RT-PCR. All data were normalized to β-actin, a house keeping gene and expressed as fold increase. Data shown represent the mean ± SD of three independent experiments. (B), (i) A549 cells were transfected with 2 μg of 5'PPP-RNA for the indicated times, and RIG-I expression was analyzed by immunoblot. (ii) A549 cells were mock-transfected or transfected with 2 μg of 5'PPP-RNA, CIAP-RNA or chemically synthesized OH-RNA for 24 hr. RIG-I expression was analyzed by immunoblot. (iii) A549 cells grown on cover-slips were mock-transfected or transfected with 2 μg of 5'PPP-RNA or CIAP-RNA for 24 hr. Cells were then paraformaldehyde fixed and immunostained with anti-RIG-I antibodies. Alexa fluor 594 goat anti-rabbit IgG Antibody (red fluorescence) or Alexa 488 (green fluorescence) were used as secondary antibodies. Nuclei were stained with Hoechst (blue fluorescence). (C) RNA was isolated from A549 cells mock transfected or trasfected with 5'PPP-RNA or CIAP-RNA for 24 hr and then infected with wild-type H5N1 virus for 24 hr (as described in Figure legend 1). Real-time RT-PCR was performed to analyze the expression of (i) IFNβ, (ii) RIG-I and (iii) NS1. All data were normalized to β-actin, a house keeping gene and expressed as fold increase. Data shown represent the mean ± SD of three independent experiments.