HMGA2-mediated transcriptional activation is dependent upon basic residues within the second AT-hook

<p><b>Copyright information:</b></p><p>Taken from "The second AT-hook of the architectural transcription factor HMGA2 is determinant for nuclear localization and function"</p><p></p><p>Nucleic Acids Research 2007;35(6):1751-1760.</p><p>Published online 25 Feb 2007</p><p>PMCID:PMC1874589.</p><p>© 2007 The Author(s)</p> () Diagram of luciferase reporter gene under the transcriptional regulation of the cyclin A promoter and the HMGA2 wild type and mutants expressed by the vectors used. () CHO cells were transiently cotransfected with 1 µg of the CycA luciferase reporter plasmid (bars 1–4) and with 3 µg of HMGA2 wt-, mS5- and mT6- EGFP expression vectors (bars 2–4 respectively). 0.1 µg of pRL-CMV Renilla luciferase expression vector was included to normalize for transfection efficiencies. Values are reported as relative luciferase activity. Standard deviations are indicated for experiments repeated three times. The amount of HMGA2 wt-, mS5- and mT6-EGFP expression was assayed by Western blot analysis using a polyclonal α-HMGA2 antibody. The subcellular localization of the expressed proteins in CHO cells was confirmed by confocal microscopy (data not shown)

Identification of HMGA2 nuclear localization signal by deletion mutagenesis

<p><b>Copyright information:</b></p><p>Taken from "The second AT-hook of the architectural transcription factor HMGA2 is determinant for nuclear localization and function"</p><p></p><p>Nucleic Acids Research 2007;35(6):1751-1760.</p><p>Published online 25 Feb 2007</p><p>PMCID:PMC1874589.</p><p>© 2007 The Author(s)</p> () Schematic representation of HMGA2 deletion mutants; each deletion mutant is fused to β-gal at the N-terminus, and to GFP at the C-terminus. Summary of intracellular localization is indicated at the right. () NIH-3T3 cells were transfected with HMGA2 deletion mutants constructs, and fusion proteins were visualized by confocal laser microscopy. Propidium iodide (PI) staining of the same nuclei is shown. At least one hundred cells per transfection were analyzed in three different experiments. Bars, 10 μm. () Western blot analysis performed with an anti-GFP antibody

Basic residues of the second AT-hook of HMGA2 are responsible for nuclear localization of fusion proteins

<p><b>Copyright information:</b></p><p>Taken from "The second AT-hook of the architectural transcription factor HMGA2 is determinant for nuclear localization and function"</p><p></p><p>Nucleic Acids Research 2007;35(6):1751-1760.</p><p>Published online 25 Feb 2007</p><p>PMCID:PMC1874589.</p><p>© 2007 The Author(s)</p> () Partial sequences of point-mutated constructs of HMGA2. Basic residues were replaced with alanine residues as indicated. Each point-mutant is fused C-terminally with EGFP (not drawn); numbers identify positions of residues on HMGA2 protein sequence. Summary of intracellular localization is indicated at right. () NIH-3T3 cells were transfected with HMGA2 point-mutant constructs, and fusion proteins were visualized by confocal laser microscopy. Propidium iodide (PI) staining of the same nuclei is shown. At least one hundred cells per transfection were analyzed in three different experiments, and at least one hundred cells per transfection were subjected to fluorescence quantification. Bars, 10 μm. () For each point-mutant construct, nuclear and cytoplasmic fluorescence were quantified, and percentage of nuclear localization was calculated. Bars indicate percentage ± standard deviation. () The expression and integrity of the fusion proteins were analyzed by Western blot using anti-GFP antibody

Additional file 1 of EMID2 is a novel biotherapeutic for aggressive cancers identified by in vivo screening

Supplementary Material 1: Table S1. Composition of the pilot AAV9 poo