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. 2012 Aug 1;23(15):2845–2855. doi: 10.1091/mbc.E12-02-0097

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© 2012 Senga et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 5: — The small GTPase Rab25, a target of Grhl2, is involved in the enlargement of a central lumen induced by Grhl2. (A) A schematic view for the identification of Rab25. The consensus DNA sequence for Grhl2 binding (AACCGGTT) was searched in the database for human and mouse genes. More than 1000 genes were found to contain the sequence within 1000 base pairs upstream of the transcription initiation site. Among those genes, Rab25 was selected as a Grhl2 target since it was up-regulated in P1 cholangiocytes (shown by the microarray result comparing neonatal cholangiocytes with hepatoblasts) and might be involved in the intracellular localization of Cldn4. (B) A potential Grhl2-binding site in the promoter region of Rab25 gene is necessary for up-regulation of Rab25 mRNA by Grhl2. Luciferase activity is detected in the presence of Grhl2, whereas the deletion of a possible binding site (GBE) diminishes the expression of luciferase. GBE, grhl2-binding element. (C, D) Overexpression of Rab25 induces the enlargement of a central lumen. (C) Representative cysts derived from HPPL and HPPL-Rab25. A cyst derived from HPPL-Rab25 has a central lumen surrounded by F-actin bundles (green) larger than the control HPPL. (D) Dot plot with bars of mean ± SEM for a representative culture, indicating that HPPL-Rab25 increased the size of lumen with statistical significance. HPPL and HPPL-Rab25 were cultured for 7 d, and the diameter of the central lumen was measured for >50 cysts per culture. Experiments were repeated four times independently. Bar, 50 μm. (E, F) Inhibition of Rab25 by a dominant-negative form of Rab25 in HPPL-Grhl2 resulted in decreased lumen size. (E) Representative cysts derived from HPPL-Grhl2 and HPPL-Grhl2+Rab25. Cysts derived from HPPL-Grhl2+dnRab25 have a central lumen smaller than HPPL-Grhl2. (F) Dot plot with bars of mean ± SEM for a representative culture, indicating that dnRab25 reduces the size of the central lumen with statistical significance. HPPL-Grhl2 and HPPL-Grhl2+dnRab25 were cultured for 7 d. The diameter of the lumen was measured >50 cysts per culture. Experiments were repeated for four times independently. Bars, 100 μm.

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