4 research outputs found

    Lithium-mediated inhibition of GSK3β leads to β-catenin accumulation and increase in <i>MENA</i> mRNA levels.

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    <p>(A) Western blot analysis of Huh7 cells treated with either 25 mM NaCl or 25 mM LiCl for indicated timepoints. RT-PCR (B) and qRT-PCR (C) analysis of Huh7 cells treated with either 25 mM NaCl or 25 mM LiCl for indicated timepoints. (D) RT-PCR analysis of U373, Sk-Mel-103 and Sk-Mel-19 cells treated with LiCl for 24 hrs. Each image is a representative of at least two independent experiments.</p

    Overexpression of β-catenin-S33Y and Wnt ligans leads to increase in <i>MENA</i> transcription.

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    <p>(A) β-catenin-S33Y mutant was stably overexpressed in Huh7 cells and <i>MENA</i> and Axin2 mRNA levels were analyzed using qRT-PCR. (B) β-catenin-S33Y was transiently overexpressed (48 hrs) in U373 cells and <i>MENA</i> and β-catenin mRNA levels were compared using RT-PCR. (C) Wnt1 or Wnt3a ligans were overexpressed in Huh7 cells (48 hrs) and <i>MENA</i> mRNA levels were compared using RT-PCR.</p

    <i>Ena</i> knock-down induces tumor formation and metastasis <i>in vivo</i>.

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    <p><i>Ena</i> (A), <i>white</i> (B) and <i>axin</i> (C) knock-down was confirmed by RT-PCR. Actin was used as an internal control. Significant increase of tumor formation (D and E) and metastasis (F and G) in eyeful (Eyf) (D, F) and sensitized (St) (E, G) flies upon knock-down of <i>ena</i> and <i>axin</i> genes is observed, when <i>white</i> knock-down is used as a control. (* indicates p<0.05; *** indicates p<0.001 as analyzed by the chi-square test). Representative images of tumor and metastasis formations in eyeful and sensitized flies upon knock-down of <i>Ena</i> (H-O). (H and I) ey-GAL4, UAS-Dl, eyeful/+; UAS-EnaRNAi/+ (“+”: wild-type chromosome) flies showing tumors in the eye tissue. Every fold in the eye tissue is counted as a single tumor. (J and K) ey-Gal4, UAS-Dl/+, UAS-EnaRNAi/+ flies with tumors in the eye. (L and M) ey-GAL4, UAS-Dl, eyeful/+; UAS-EnaRNAi/+ flies showing metastasis on the abdomen and thorax. (N and O) ey-Gal4, UAS-Dl/+, UAS-EnaRNAi/+ flies with metastasis on the back and abdomen.</p

    Promoters of mammalian <i>MENA</i> orthologs have functional Tcf4-binding elements (TBEs) that are regulated by the Wnt/β-catenin pathway.

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    <p>(A) Schematic representation of the promoters and the identified putative TBEs. The location of the TBEs is indicated by boxes and the sequences and locations with respect to transcriptional start sites (denoted by arrows) are shown under the boxes. Predicted binding motifs for other transcription factors are also indicated. The length of the bar is 100 bp. The WWCAAWG sequence is the consensus sequence for TBEs. (B) Alignment of proximal (TBE#1) and distal (TBE#2) TBE sequences. (C, D and E) Huh7 cells or HeLa cells were transfected with pGL3-<i>MENA</i> reporter plasmid and the promoter activity was stimulated by co-transfecting degradation-resistant β-catenin, Tcf4 or Wnt ligans or inhibited by co-transfecting dominant-negative Tcf4 (ΔNTcf4). The bars are given as averages (n = 3) normalized to Renilla luciferase activity, which was used an internal control. Error bars represent standard deviation (* = p<0.05, ** = p<0.01 and *** = p<0.005, Student's t-test). Each graph is a representative of at least two independent experiments. “β-cat” denotes the quadruple-mutant β-catenin-4m (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037013#s4" target="_blank">Materials & Methods</a>). (F) Chromatin immunoprecipitation assay was done with monoclonal anti-β-catenin antibody and PCR (35–40 cycles) was used to detect <i>MENA</i> promoter fragments. β-catenin was found to interact with <i>MENA</i> promoter in Huh7 and HEK293T cell context, as well as in mouse brain tissue and liver tissue contexts. All experiments were repeated at least twice. An unrelated monoclonal antibody was used as the control IgG. The lower bands that appear in the mouse brain gel image are the primer-dimer bands. The arrow indicates the band of the expected size.</p
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