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. 2012 Aug 1;23(15):2856–2866. doi: 10.1091/mbc.E10-12-1010

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© 2012 Demelash 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: — p35 is a direct target of hASH1, regulates migration in human SCLC cell line, and is coexpressed with hASH1 in primary SCLC tumor. (A) Photomicrograph of increased expression of p35 in migrating DMS53 SCLC cells. The most intense immunoreactivity is seen in groups of three to five cells (blue asterisk) that are migrating away from the main colony (circle), which is composed of cells with rounder shapes that appear minimally positive. (immunoperoxidase stain). (B) Quantification of migration (bar graph) of DMS53 SCLC cancer cells in scratch assay. Migration was significantly reduced by the CDK5 inhibitor roscovitine (p < 0.001), even at 6 h. (C) Photomicrographs of nuclear hASH1 (top, left: brown, immunoperoxidase) and p35 (top, right: green) immunofluorescence in migrating DMS53 cells. Colocalization (bottom) of nuclear hASH1 (pseudowhite) in several polarized, migrating DMS53 cells that are also positive for p35 (green; immunoperoxidase and immunofluorescence double labeling). (D) ChIP assay indicates direct binding of hASH1 (ASH1) to the CDK5 activator p35. (E) Photomicrograph of coexpression of nuclear hASH1 (ASH1, red) with p35 (green) in a human SCLC tumor sample. Most cells express both hASH1 and p35 (double immunofluorescence).