PMC

SIRT1 regulates Wnt target gene expression. (A) HCT116 cells were treated with either vehicle control (DMSO) or 50 μM or 100 μM cambinol (SIRT1 inhibitor) for 24 h, and total RNA was isolated and quantitative RT-PCR analysis of Wnt target genes (BMP4, cMyc, and cyclin D1) was performed using intron-spanning primers. (B) T-47D or HCT116 cells were treated with either vehicle control (DMSO) or 50 μM or 100 μM cambinol (SIRT1 inhibitor) for 18 h, and total RNA was isolated and semiquantitative RT-PCR analysis of Wnt target genes (BMP4 and cyclin D1) and β-actin was performed using intron-spanning primers. (C) T-47D and HCT116 cells were treated with either vehicle control (DMSO) or 50 μM or 100 μM cambinol and Western blots for unphosphorylated (active) β-catenin, total β-catenin, and β-actin were performed.

SIRT1 and Dvl form a complex in vivo. (A) HEK293 cells were transiently transfected with SIRT1-Flag and either an empty vector, Dvl-1, Dvl-2, or Dvl-3. SIRT1-Flag was immunoprecipitated and Western blots for each Dvl family member or SIRT1 was performed. (B) (Upper) Interaction between endogenous SIRT1 and Dvl-3 in T-47D cells was performed by immunoprecipitation of Dvl-3 followed by blots for Dvl-3 and SIRT1. (Lower) SIRT1 was immunoprecipitated followed by blots for Dvl-3 and SIRT1. (C) (Upper) HCT116 cells were harvested, endogenous SIRT1 was immunoprecipitated, and Dvl-1 and SIRT1 were analyzed by Western blot. (Lower) SIRT1 was immunoprecipitated followed by blots for CK2α and SIRT1. (D) The protein expression patterns of active and total β-catenin were analyzed by Western blotting in the indicated lines. (E) Either control IgG or β-catenin was immunoprecipitated followed by blots for β-catenin and SIRT1.

SIRT1 regulates transient Wnt signaling. (A) Total RNA was isolated from four colon cancer lines (HT-29, HCT116, RKO, and DLD-1) and the relative levels of three candidate Wnt genes (Wnt3a, Wnt5b, and Wnt7a) were analyzed by quantitative RT-PCR analysis. (B) HCT116 cells were analyzed in a transwell migration assay in the absence or presence of purified Wnt3a ligand (260 ng/mL) ± cambinol (130 μM). (C) Changes in migration were measured after 8 h and cells were manually counted in a (680 × 512) still image field with an Olympus DP 70 microscope. Three assays were counted with duplicates per condition.

Dishevelled expression is regulated posttranslationally. (A) Total RNA was isolated from the indicated cell lines and RT-PCR analysis of the three Dishevelled (Dvl) genes was performed using intron-spanning primers. For each amplification, either the reverse transcriptase was excluded (−) or included (+) in the reverse-transcription reaction and β-actin was included as a control. (B) The protein expression patterns of all three Dvl family members, SIRT1, and β-actin were analyzed by Western blotting. (C) T-47D, HEK293, and DLD-1 cells were treated with vehicle control (dimethyl sulfoxide; DMSO) or 50 μM or 100 μM cambinol (SIRT1 inhibitor) for 18 h (T-47D) or 24 h (HEK293 and DLD-1) and the indicated proteins were analyzed by Western blot.

SIRT1 inhibition leads to a significant reduction in Dvl protein expression. (A) T-47D cells were transfected for 48 h with either control vector (pRetroSuper) or with plasmids encoding two shRNAs specific for SIRT1 (589 or 1091). Expression of Dvl-2 and Dvl-3 was assessed by Western blot. (B) Control or SIRT1 shRNAs were transfected into HEK293 cells and Dvl-3 was analyzed by Western blot. The 1091 shRNA is effective at reducing SIRT1 protein levels and causes a consistent reduction in Dvl-3 protein levels. (C) Control or SIRT1 siRNAs were transfected into DLD-1 cells for 24 h and Dvl-2 and Dvl-3 were analyzed by Western blot. (D–F) T47-D cells were transfected with (D) control siRNA and SIRT1 siRNA, (E) SIRT2 siRNA, or (F) both SIRT1 and SIRT2 siRNA at 100 nM for 72 h to measure Dvl-2, Dvl-3, SIRT1, and/or SIRT2 expression via Western blot. (G) T-47D cells were pretreated with 5 μM lactacystin (Lacta) (left) or 0.5 μM MG132 (right) for 15 min followed by a 22-h treatment with or without 100 μM cambinol. DVL2, DVL3, and actin levels were measured. Control blots for a polyubiquitinated protein (p27) and a loading control (β-actin) were performed on the same membrane.