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Fig. 4. FoxO regulates Rdh expression in mouse RPE.
(A) Genomic DNA sequences of mouse Adh1, Adh7, and Rdh10 were compared with those of other vertebrate homologous genes and the consensus sequences that can be recognized by FOXO in the upstream of transcription initiation sites are marked by yellow asterisks (see details in Materials and Methods section). (B) pGL3 luciferase reporter constructs containing the upstream sequences of mouse Adh1, Adh7, and Rdh10 genes were expressed in 293T cells and the effects of co-expressed AKT1-CA and AKT1-KM on the luciferase expression were examined. (C) DNA fragments that were pulled down from 6 months-old mouse RPE by ChIP with rabbit anti-FOXO3a IgG or pre-immune rabbit IgG. Relative amounts of the upstream sequences of Adh1, Adh7, and Rdh10, which include the FoxO target sequences, in the ChlPed DNA fragments were investigated by RT-qPCR. Numbers of samples analyzed are shown in the bars of each graph. Error bars denote SD. *P < 0.05, **P < 0.01, ***P < 0.001. (D) The pGL3 luciferase reporter constructs were co-expressed with wild-type FOXO3A or FOXO3A-TM mutant, which is resistant to Akt-mediated phosphorylation, and the effects of those FOXO3A variants on the luciferase expression were examined. Error bars denote SD. n.s., not significant. (E) Relative amounts of FoxO1, FoxO3a, FoxO4, pFoxO1(Thr24)/pFoxO3a(Thr32), and pAkt(T308) in P21 TRP1-Cre and Pten-cko littermate mouse RPE were investigated by WB with the antibodies detecting the corresponding proteins. (F) Subcellular distribution of FoxO3a in P21 TRP1-Cre and Pten-cko mice RPE was investigated by immunostaining. Nuclei and F-actin in mouse RPE was visualized by staining with DAPI (blue) and phalloidin-Alexa647 dyes (green), respectively. Scale bar = 50 µm.
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