New Reagent for Highly Efficient Synthesis of Trifluoromethyl-Substituted Arenes and Heteroarenes

A new reagent trimethylsilyl chlorodifluoroacetate (<b>TCDA</b>) is reported for the introduction of a −CF₃ group to arenes and heteroarenes. Compared with current known reagents, <b>TCDA</b> shows very broad scope with respect to electron-deficient, -neutral, and -rich aryl/heteroaryl iodides as well as excellent functional group tolerance, including ester, amide, aldehyde, hydroxyl, and carboxylic acid

Electrophoretic mobility shift assay (EMSA) confirms that SRF protein binds to SIRT2 promoter sequence containing the classic CArG box (CCATAATAGG).

<p>To ensure that it is the SRF protein that binds to hSIRT2 CArG sequence, in-vitro synthesized SRF protein was used in the binding assay. EMSA indicates that purely synthesized SRF protein binds to classic CArG box (32P-CArG, lane b), but SRF does not bind to CArG-like sequence (CAATAAAAGG)(lane E) in which a “C” is substituted with a “A”. The arrow indicates the binding activity of full-length of SRF protein to the classic CArG sequence, which is at the similar position as what was observed in our previous experiments [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190011#pone.0190011.ref033" target="_blank">33</a>].</p

Altered expression of miR* during adult aging.

<p>Six miR*s that were differentially expressed in old (O) vs young adult (YA) hearts.</p

SIRT2 gene is regulated by Rho/SRF signaling pathway and SRF cofactors.

<p><b>6A.</b> SIRT2 expression in response to serum deprivation did not change after 6 hours of CCG-1423 treatment (1.1+0.08, P>0.05 NS, n = 3), but was significantly inhibited after 24 hours of CCG-1423 treatment (0.2+0.09, p< 0.01**, n = 3), indicating that the Rho/SRF signaling pathway regulates SIRT2 gene expression. <b>6B.</b> The SIRT2 expression was repressed by both SRF (0.57+0.06, p<0.05*, n = 3) and p49 (0.7+0.07, p<0.05*, n = 3) under normal culture condition without stress, suggesting that SRF and its cofactors regulate expression of the SIRT2 gene.</p

The miRNAs that were decreased during adult aging.

<p>Thirty-one miRNAs were decreased in old (O) vs. young adult (YA) hearts.</p

The 32 miRNAs and their corresponding miRNA gene clusters.

<p>Approximately half of the miRNAs (32 out of 65 miRNAs) that were differentially expressed in the old heart belong to 11 miRNA gene clusters. These 32 miRNAs had greater than 1.5-fold change in old (O) versus young adult (YA) hearts. Other miRNAs in the clusters with less than 1.5-fold change in expression are not shown in this table.</p

The response of SIRT2 mRNA expression to serum deprivation and serum restoration following serum deprivation.

<p><b>5A.</b> The Hela cells were initially maintained in DMEM medium (glucose concentration: 4.5 g/L) containing 10% FBS. After washing with PBS twice, the cells were subjected to serum deprivation stress with DMEM containing 0.1% FBS for 3 hours, 6 hours, 24 hours and 48 hours, respectively. SIRT2 expression is increased from 24h to 48h after serum deprivation. <b>5B.</b> For the treatment of serum restoration following serum starvation, the cells were first cultured in DMEM containing 0.1% serum for 48 hours, then were cultured in DMEM containing 10% serum for additional 6, 18, 24, and 48 hours, respectively. SIRT2 expression was decreased after 6 hours serum restoration (0.15+0.08, p<0.01**, n = 3), 18 hours serum restoration (0.15+0.07, p<0.01**, n = 3), 24 hours serum restoration (0.018+0.06, p<0.01**, n = 3) and 48 hours serum restoration (0.29+0.09, p<0.01**, n = 3).</p

SRF and SRF-binding proteins regulate SIRT2 gene promoter activity.

<p>SRF upregulated the SIRT2 gene promoter activity by 1.39 fold + 0.051 (p< 0.05*, n = 3); SRF and myocardin synergistically activated SIRT2 gene promoter activity by 1.97 fold + 0.079 (p< 0.01**, n = 3); while p49/STRAP repressed the SIRT2 promoter activity (1.26 fold + 0.053, p< 0.05*, n = 3) that was induced by myocardin.</p

The miRNA gene clusters and their genomic structures of cluster #1∼cluster #4, which are part of 11 miRNA gene clusters shown in figure 5, 6 and 7.

<p>The number of miRNA genes in each cluster ranged from 2 to 71. In 10 out of 11 clusters, the expression of most miRNA genes in that cluster was in the same direction (the arrow <b>↑</b> for increased expression, and the arrow <b>↓</b> for decreased expression). Please note that part of the miRNAs with changes in expression greater than 1.5 fold in old (O) vs. young adult (YA) hearts were listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034688#pone-0034688-t002" target="_blank">table 2</a>.</p

The effect of Ago1 and Ago2 on the expression of miR-21*.

<p>In the transfection assays, the cells were transfected with empty vector (Control), or Ago1, Ago2, mir-21, Ago1 and mir-21 as well as Ago2 and mir-21. The miR-21* passenger strand was upregulated by Ago1, Ago2, mir-21 gene, Ago1 and mir-21 as well as Ago2 and mir-21 gene. The data indicated that Ago1 and Ago2 synergistically upregulated miR-21* passenger strand when mir-21 gene was increased. # refers to p<0.05, n = 3. ## refers to p<0.01, n = 3.</p