Figure 2. Down-regulation of DDX1 facilitates AMD.

(A) HeLa-TO cells were transfected with a construct expressing GB-ARE^GMCSF mRNA under the control of a Tet-regulatory promoter and a construct constitutively expressing GB-GAPDH mRNA under the control of the CMV promoter. The cultures were also transfected a control siRNA (CAT) or a DDX1 siRNA. Cytoplasmic RNA was isolated at different time points after the addition of doxycycline (Dox). The levels of GB-ARE^GMCSF and GB-GAPDH mRNAs were analyzed by Northern blot. Signals of GB-ARE^GMCSF mRNA were quantified by a phosphorimager and normalized to that of GB-GAPDH mRNA. The calculated half-lives (t_1/2; n=3) of GB-ARE^GMCSF mRNA are shown as mean values ± standard deviations from three independent experiments. P value is indicated and calculated by Student’s t-test using Microsoft Excel software. (B) Downregulation of DDX1 by siRNA. Extracts of HeLa-TO cells in (A) were subjected to immunoblot analysis with anti-DDX1 or anti-HuR antibodies. Different amounts of CAT siRNA-treated extracts (12, 25, 50, or 100% of the amounts used in lane 5) were loaded to estimate knockdown efficiency.