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SIRT1 inactivation inhibits Wnt and promotes p53 a pathway.

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posted on 14.06.2013 by Vid Leko, Gemma J. Park, Uyen Lao, Julian A. Simon, Antonio Bedalov

A) Representative photomicrographs showing β-catenin immunohistochemical staining of intestinal sections from APC+/min SIRT1+/+ and APC+/min SIRT1−/− mice. Scale bar indicates 50 µm length. Polyps from both groups demonstrate intense cytoplasmic and nuclear staining for β-catenin. B) Bar graph with frequencies of crypts with indicated number of cells with nuclear β-catenin staining in normal appearing mucosa of APC+/min SIRT1+/+ and APC+/min SIRT1−/− mice. The average number of β-catenin positive cells per crypt is reduced in APC+/min SIRT1−/− mice compared to APC+/min SIRT1+/+ animals 2.1±1.2 vs 3.2±1.3 (p = 1.5×10−5, at least 100 crypts per genotype were scored). C) Representative photomicrographs of normal appearing mucosa from APC+/min SIRT1+/+ and APC+/min SIRT1−/− mice demonstrating a reduced number of basal cells with nuclear β-catenin in APC+/min SIRT1−/− animals. Arrows are directed toward representative basal cells with nuclear β-catenin. Scale bar indicates 25 µm length. D) Inhibition of SIRT1 with EX527 (2 µM) and cambinol (50 µM) reduces activity of the TCF/LEF driven firefly luciferase reporter (TOP FLASH) transiently transfected into SW480 cells. TOP FLASH luciferase reporter contains minimal promoter along with three TCF binding sites, which have been mutated in FOP FLASH reporters. Bars represent means ± SEM of relative firefly luciferase activity normalized to renilla luciferase activity from the thymidine kinase promoter-driven renilla reporter that was co-transfected with TOP FLASH and FOP FLASH reporters. Each transfection is carried out in quadruplicate. **p<0.01. E) SW480 cells with shRNA-mediated downregulation of SIRT1 (top: western blot for SIRT1 and actin) exhibit reduced activity of the transiently transfected TCF/LEF driven firefly luciferase reporter (TOP FLASH). Bars represent means ± SEM of relative firefly luciferase activity normalized to renilla luciferase activity from the thymidine kinase promoter-driven renilla reporter that was co-transfected with TOP FLASH and FOP FLASH reporters. Each transfection is carried out in quadruplicate. *p<0.05. F) Immunoblot for acetyl-p53, p53 and actin from cells treated with etoposide, SIRT1 inhibitor EX527 or the combination of the two drugs. Inhibition of SIRT1 leads to p53 hyperacetylation in Hct116 colon cancer cell line. Hyperacetylation of p53 is observed in cells treated with EX527 and a combination of EX527 and etoposide. Etoposide alone modestly induces p53 acetylation.

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