β-catenin knockdown recapitulates the affect of SIRT1/2 inhibition on FZD7.

<p>T-47D cells were treated for 24 hrs with siRNA against β-catenin (<b>A</b>) or FZD7 (<b>B</b>). Expression of related genes (β-catenin, FZD7, and GAPDH) were measured by real-time PCR. Gene expression was normalized to GAPDH. <b>B</b>. T-47D cells were transfected with siRNA against either β-catenin or FZD7 for 48-hours using oligofectamine, cells were processed for Western blot analysis probing for β-catenin and FZD7. An * indicates a p-value <0.05 versus control. Samples were normalized to non-immune IgG, (n = 3).</p

Pharmacologic inhibition of SIRT1/2 decreases FZD7 mRNA expression in both MDA-MB-231 and T-47D cells.

<p><b>A</b>. MDA-MB-231 and T-47D cells were treated with 50 and 100 µM cambinol for 24 hrs. RNA was isolated from cells for cDNA synthesis. RT-PCR was performed to quantitate gene expression changes. <b>B</b>. MDA-MB-231 (upper) cells and T-47D (lower) cells were treated with 50 and 100 µM cambinol for 24 hrs. Cells were harvested and RNA isolated for analysis of mRNA via quantitative real-time PCR. <b>C</b>. MDA-MB-231 cells were treated with cambinol for 24 hrs. Following drug application, cells were lysed in RIPA buffer for protein analysis. <b>D</b>. and <b>E</b>. RNA was harvested from MDA-MB-231 or T-47D cells (respectively) that had been treated for 18 hrs with inhibitor VII, and analyzed using semi-quantitative PCR techniques. <b>F</b>. MDA-MB-231 cells were treated with inhibitor VII for 18 hrs, and then lysed in RIPA buffer for protein analysis. <b>G</b>. T-47D cells were treated the same as MDA-MB-231 cells with inhibitor VII for 18 hrs, and then protein was analyzed via Western blot. Data plotted are from average of 3 experiments +/− S.E.M, * p-value <0.05.</p

List of PCR primers.

<p>List of PCR primers.</p

SIRT1/2 activity and FZD7 expression are critical to cell motility and growth.

<p><b>A</b>. MDA-MB-231 cells were transfected for 24 hrs with siRNA against FZD7 and protein expression of FZD7 was later determined by Western blot analysis. <b>B</b>. MDA-MB-231 cells that had been transfected with FZD7-specific siRNA 24 hrs were placed on 96-well plates for wound healing/migration assay. Wound healing was performed for total 72 hrs to allow complete wound closure. <b>C</b>. Following 24 hrs transfection of MDA-MB-231 cells with FZD7-specific siRNA, cells were plated in triplicate to measure growth rates over 72 hrs. Each point plotted is the average of 3 independent experiments. <b>D</b>. MDA-MB-231 cells were treated with SIRT1/2 inhibitor VII at 50 µM, and placed on transwell plates for migration analysis. Migration was conducted for 14 hrs. Transwells were then washed and stained with crystal violet before visualization on microscope. Bar graph depicts 3 experimental replicates and error bar is the S.E.M. An * indicates a p-value <0.05 versus control.</p

Variable frizzled receptor expression in breast cancer cell lines.

<p><b>A</b>. Semi-quantitative RT-PCR analysis of frizzled receptors 1–9 to determine expression pattern in MDA-MB-231 cells and T-47D cells. Samples were resolved on 1% low melting agarose gel.</p

β-catenin regulation of FZD7 is sirtuin-dependent in breast cancer cells.

<p><b>A</b>. Cells were treated with 50 and 100 µM of inhibitor VII, and protein was harvested in RIPA buffer to analyze active b-catenin expression. <b>B</b>. Chromatin immunoprecipitation (ChIP) for β-catenin at the promoter of FZD7 gene in T-47D cells, followed by qPCR with primers that target the domains through -7 kb region of FZD7 promoter/enhancer (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098861#pone-0098861-t001" target="_blank">Table1</a>). <b>C</b>. ChIP analysis of c-Jun enrichment at the FZD7 promoter/enhancer. T-47D cells treated with inhibitor VII or vehicle for 24 hrs and then ChIP performed as previously described. <b>D</b>. ChIP analysis of Dvl1 enrichment at the FZD7 promoter/enhancer. <b>E, F and G</b>. T-47D cells were treated with 100 µM of inhibitor VII for 18 hrs. Cells were then harvested for ChIP-qPCR analysis. Data plotted are from average or 4 experiments +/− S.E.M. An * indicate a p-value <0.05 versus IgG sample, A ** indicates a p-value <0.05 versus DMSO treated samples. Samples were normalized to non-immune IgG, (n = 4).</p