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

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

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

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

Phospholipase D1 Couples CD4⁺ T Cell Activation to c-Myc-Dependent Deoxyribonucleotide Pool Expansion and HIV-1 Replication

<div><p>Quiescent CD4+ T cells restrict human immunodeficiency virus type 1 (HIV-1) infection at early steps of virus replication. Low levels of both deoxyribonucleotide triphosphates (dNTPs) and the biosynthetic enzymes required for their <i>de novo</i> synthesis provide one barrier to infection. CD4+ T cell activation induces metabolic reprogramming that reverses this block and facilitates HIV-1 replication. Here, we show that phospholipase D1 (PLD1) links T cell activation signals to increased HIV-1 permissivity by triggering a c-Myc-dependent transcriptional program that coordinates glucose uptake and nucleotide biosynthesis. Decreasing PLD1 activity pharmacologically or by RNA interference diminished c-Myc-dependent expression during T cell activation at the RNA and protein levels. PLD1 inhibition of HIV-1 infection was partially rescued by adding exogenous deoxyribonucleosides that bypass the need for <i>de novo</i> dNTP synthesis. Moreover, the data indicate that low dNTP levels that impact HIV-1 restriction involve decreased synthesis, and not only increased catabolism of these nucleotides. These findings uncover a unique mechanism of action for PLD1 inhibitors and support their further development as part of a therapeutic combination for HIV-1 and other viral infections dependent on host nucleotide biosynthesis.</p></div

PLD1 activity is required for normal expression of nutrient transporters in activated T cells.

<p>(A) Cell surface expression of glucose transporter Glut1 and activation marker CD25 as determined by flow cytometry on resting primary CD4+ T cells (shaded histogram) or after stimulation with anti-CD3/anti-CD28 beads for 48h in the absence (solid line) or presence of PLD1i (dashed line). (B) Western blot analysis of protein expression in whole cell lysates from primary CD4+ T cells treated as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004864#ppat.1004864.g001" target="_blank">Fig 1A</a>. (C) Q PCR analysis of expression of target genes in primary CD4+ T cells nucleofected with siRNA and then stimulated for 24 h as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004864#ppat.1004864.g001" target="_blank">Fig 1C</a>. T cells were nucleofected with non-targeting (NT) (black bars), c-Myc (grey bars), or PLD1 (open bars)-specific siRNAs. mRNA abundance in NT siRNA sample was set to1. Error bars represent standard error from the mean of triplicate samples. Data are representative of experiments from three independent donors.</p

Inhibition of PLD1 activity limits proliferation of activated CD4+ T cells.

<p>(A) Schematic showing the different cell cycle phases detected by staining DNA and RNA with 7-AAD and Pyronin Y, respectively. (B) Cell cycle analysis of primary CD4+ T cells after 72h stimulation with anti-CD3/anti-CD28 beads in the presence or absence of 10 μM PLDi by staining with Pyronin Y and 7-AAD followed by flow cytometry. (C) Flow cytometric proliferation analysis of CellTRACE Violet-labeled CD4+ T cells pretreated with 50 μM of Myci, 5 or 10μM of PLDi after stimulation with anti-CD3/anti-CD28 beads for 72h in the presence or absence of inhibitors. The number of cellular divisions present in treated cultures is indicated above each peak.</p

PLD1 inhibition decreases the c-Myc-dependent dNTP biosynthetic transcriptional program.

<p>(A) Western blot analysis of protein expression in whole cell lysates prepared from resting primary human CD4+ T cells that were pretreated for 24h with DMSO vehicle, 10 or 5 μM of PLD1i, 50 or 25 μM of c-Myci, 10 μM U0126, or 100 nM of rapamycin. Cells were either left resting or stimulated with 5μg/ml PHA and 20 U/ml IL2 in the continued presence of DMSO or inhibitors for 48h before cell harvest. (B) RRM2 mRNA levels in resting primary CD4+ T cells pretreated for 24h with DMSO vehicle, 100 μM c-Myci, or 10 μM PLDi, then left resting or stimulated as in (A) as determined by quantitative real-time PCR. mRNA abundance in mock controls was set to1. Error bars represent standard error from the mean of triplicate samples. Data are representative of experiments from three independent donors. UD; undetectable levels of mRNA. (C) Resting primary human CD4+ T cells were transfected via nucleofection with siRNAs targeting PLD1 or c-Myc. Cells were allowed to recover for 24 h and then stimulated for 48 h with anti-CD3/anti-CD28 beads, harvested and western blot analysis performed as in (A). (D) In activated CD4+ T cells, PLD1 is shown to regulate c-Myc through ERK or mTOR-dependent mechanisms. This results in the increased expression of genes essential for uptake of amino acids (box 1), glucose (box 2), and synthesis of nucleotides (box 3).</p