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SIRT1 inhibits FOXA2-driven transcription.

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posted on 2014-05-29, 02:57 authored by Rogier van Gent, Claudio Di Sanza, Niels J. F. van den Broek, Veerle Fleskens, Aukje Veenstra, Gerdine J. Stout, Arjan B. Brenkman

(A) and (B) SIRT1 co-transfection affects the FOXA2 transcriptional program. HEK293T cells were co-transfected with the indicated constructs and TK Renilla in combination with (A) G6pase and (B) CPT1a reporter constructs. After 48 hours, a luciferase-driven transcriptional assay was performed to assess FOXA2-mediated transcriptional activity on the promoter constructs. (C) SIRT1 knock-down increased FOXA2-driven transcription on the G6pase gene promoter. HEK293T cells were transfected with either control, or SIRT1 siRNA and subsequently co-transfected as in (A). Transcriptional activity was assessed via luciferase assays. (D) SIRT1 affects FOXA2-driven transcription by reducing FOXA2 protein levels. HEK293T cells were transfected as in (A) with FLAG-FOXA2 alone or in combination with increasing amounts of MYC-SIRT1. Empty pcDNA3 vector was co-transfected to balance between samples for the amount of DNA transfected. Transcriptional analysis was determined by luciferase assays. Values shown represent the mean of three independent experiments +SEM. Statistical analysis was performed by two-tailed Student's t-Test. **, P<0.01. Protein levels were assessed by Western Blotting using indicated antibodies. Actin was used as loading control.