Regulation of the Foxo1 promoter by ALX3. (A) Quantitative RT-PCR of Alx3 mRNA in embryos from non-diabetic (ND) or diabetic (D) wild type mice (n = 7 per group). (B) Western blot showing ALX3 in primary MEM cells cultured in the indicated concentrations of glucose. (C) Densitometric measurements of ALX3 bands relative to actin bands from three western blots similar to that shown in B. D) Quantitative RT-PCR of Nrf2, Foxo1 or Foxo4 mRNAs, extracted from embryos of non-diabetic (ND) or diabetic (D) wild type (white bars) or Alx3-deficient (black bars) mice; n.s., non-significant (n = 7 per group). (E) ALX3-binding site and its position in the mouse Foxo1 promoter. (F) Electrophoretic mobility shift assays showing the binding of proteins from nuclear extracts of primary MEM cells to an oligonucleotide probe containing the sequence indicated in E. The absence (−) or presence of competing or nonspecific competing (NSC) oligonucleotides at the indicated fold molar excess, or of an ALX3 antibody or control IgG, is depicted on top. Arrows indicate complexes containing ALX3. (G,H) Relative luciferase activity elicited in Hela (G) or primary Alx3-defcient MEM (H) cells co-transfected with the reporter plasmids Foxo1T81Luc or control pT81Luc, and an ALX3 expression plasmid (n = 5). (I) Quantitative PCR amplification of Foxo1 chromatin immunoprecipitated with anti-ALX3 antiserum or with control non-immune rabbit serum (NRS) from primary MEM cells isolated from wild type mice and cultured in the presence of the indicated concentrations of glucose. Horizontal lines represent the mean (n = 4). (J) Electrophoretic mobility shift assays showing binding of nuclear extracts from primary MEM cells cultured at the indicated concentrations of glucose to the Foxo1 promoter site indicated in E. Arrows indicate the presence of ALX3. (K) Western blot showing FOXO1 in primary wild type (left panels) or Alx3-deficient (right panel) MEM cells cultured in the indicated concentrations of glucose. A representative example of three independent experiments with similar results is depicted. Uncropped blots are provided in Supplementary Fig. S5. Except in I, all values represent mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathop{+}\limits_{-}$$\end{document}+− s.e.m. *P < 0.05, **P < 0.01, Student’s t-test.