The overexpression of p300 increases ALP activity and mineral formation in HDPCs.

<p>(A) HDPC/V, HDPC/p300 and HDPC/p300-ΔHAT cells were cultured for 3 days in normal growth medium or odontoblastic induction medium, and the ALP activity in these cells was measured. GM, normal growth medium; IM, odontoblastic induction medium. (B) The effect of p300 on the formation of mineralized nodules in HDPCs cultured in odontoblastic induction medium, as analyzed by alizarin red S staining on day 21(×100). a: HDPC/V; b: HDPC/p300; c: HDPC/p300-ΔHAT. Scale bar, 100 µm. d: The histogram shows the quantification of mineralization by densitometry and reveals that remarkable decreases in mineralization occurred in HDPC/p300 and HDPC/p300-ΔHAT cells relative to control cells. All results are presented as the means ± SD of three independent experiments. Procedures were performed as described in the text (n = 3). * Statistically significant difference relative to the control, <i>P</i><0.05.</p

CHIP assay shows that p300 binds to the promoter region of <i>OCN</i> and <i>DSPP</i> in HDPCs.

<p>(A, B) Cells were cross-linked with formaldehyde. Chromatin was immunoprecipitated with anti-p300 or anti-H3K9Ac antibodies. The chromatin was eluted, reverse cross-linked, and the eluted DNA was analyzed by PCR.</p

p300 regulates the expression of OCT4, NANOG and SOX2, whereas HAT mutant p300 suppresses the expression of NANOG and SOX2.

<p>(A) Real-time qPCR estimation of the relative endogenous mRNA levels of <i>OCT4, NANOG</i> and <i>SOX2</i> in HDPC/p300 and HDPC/V cells. The mRNA levels of each product were normalized to the mRNA levels of GAPDH. (B) Western blotting analysis (right panel) and densitometric evaluation (left panel; expressed as the ratio of protein levels to GAPDH levels) measuring the levels of OCT4, NANOG and SOX2 proteins in HDPC/p300 and HDPC/V cells. The expression of GAPDH was used as an internal control. (C) Measurement of the relative endogenous mRNA levels of <i>NANOG</i> and <i>SOX2</i> in HDPC/V, HDPC/p300 and HDPC/p300-ΔHAT cells using real-time qPCR. The mRNA levels of each product were normalized to GAPDH mRNA levels. (D) The results were further confirmed by western blotting (right panel) and densitometric evaluation (left panel; expressed as ratio to GAPDH). GAPDH was used as an internal control. All results are expressed as the means ± SD of three independent experiments. Procedures were performed as described in the text (n = 3). * Statistically significant difference relative to the control, <i>P</i><0.05.</p

The stable overexpression of p300 and p300-ΔHAT in HDPCs.

<p>(A) real-time qPCR was performed to measure the relative levels of p300 and p300-ΔHAT mRNA after the transduction with lentiviral vectors. The level of each product was normalized to GAPDH mRNA levels. (B) Protein expression levels of p300 and p300-ΔHAT were assessed by western blotting analysis (right panel) and densitometric evaluation (left panel; expressed as ratio to GAPDH). The expression of GAPDH served as a control. All results are presented as the means ± SD of three independent experiments. Procedures were performed as described in the text (n = 3). * Statistically significant differences relative to the control, <i>P</i><0.05.</p

The overexpression of p300 regulates the expression of odontoblastic marker genes in HDPCs in normal growth medium or odontoblastic induction medium.

<p>(A) Real-time qPCR examination of the relative endogenous mRNA levels of <i>DMP-1, DSPP, DSP, OPN</i> and <i>OCN</i> in cells with overexpressed p300 and in negative control cells cultured under normal culture conditions. The mRNA levels of each product were normalized to GAPDH mRNA levels. (B) The overexpression of p300 increases the expression of odontoblastic marker genes in HDPCs induced to differentiate. Total RNA was extracted from the induced cells. The endogenous mRNA expression of <i>DMP-1, DSPP, DSP, OPN</i> and <i>OCN</i> was measured by real-time qPCR on days 7 and 14. GAPDH was used as an internal control. All results are presented as the means ± SD of three independent experiments. Procedures were performed as described in the text (n = 3). * Statistically significant difference relative to the control, <i>P</i><0.05.</p

The effect of p300 on the proliferation of HDPCs.

<p>(A) Cell growth curves of HDPC/p300, HDPC/p300-ΔHAT and HDPC/V cells were constructed with the results of the CCK8 assay. The growth curves showed that p300 does not have a significant effect on the proliferation of HDPCs. (B) The BrdU assay revealed no significant differences in the amount of DNA synthesized by HDPC/p300, HDPC/p300-ΔHAT and HDPC/V cells. Newly synthesized DNA is stained red by BrdU and nuclei are stained blue by DAPI. Procedures were performed as described in the text (n = 3). All results are presented as the means ± SD of three independent experiments.</p

TMP up-regulated SIRT1 expression.

<p>The bEnd.3 and HUVEC cells were treated with Control (5.6 mmol/L glucose, CTR), High glucose (30 mmol/L, HG), Control+ TMP (30 µmol/L) and High glucose+TMP (30 µmol/L) for 48 hrs. The mRNA levels of SIRT1 were determined by RT-PCR (<b>A</b> and <b>B</b>). Levels of SIRT1 expression were analyzed by Western blotting in bEnd.3 (<b>C</b> and <b>D</b>) and HUVEC cells (<b>E</b> and <b>F</b>). Representative blots (<b>C</b> and <b>E</b>) and quantification data (<b>D</b> and <b>F</b>) are shown. Data are mean ± SEM (n = 4). ^#<i>P</i><0.05 vs. Control; ^##<i>P</i><0.01 vs. Control; ^*<i>P</i><0.05 vs. High glucose; ^**<i>P</i><0.01 vs. High glucose.</p

TMP increased NO production in endothelial cells exposed to high glucose.

<p>DAF-FM indicator was used to detect NO levels in HUVEC treated with (<b>A</b>) 5.6 mmol/L glucose (Control), (<b>B</b>) High glucose (30 mmol/L) and (<b>C</b>) High glucose+TMP (30 µmol/L). The treated cells were photographed by fluorescence microscopy (<b>A–C</b>). (<b>D</b>) bEnd.3 and (<b>E</b>) HUVEC were treated as indicated for 48 hrs, and NO production was measured using flexstation3 and quantified with the changes in fluorescence intensity. “F0” represents fluorescence of Control group and “F” depicts fluorescence of other treated groups. Data are mean ± SEM (n = 4). ^#<i>P</i><0.05 vs. Control; ^##<i>P</i><0.01 vs. Control; ^*<i>P</i><0.01 vs. High glucose.</p

Vasorelaxant effects of TMP on rat aortic rings.

<p>The freshly isolated rat aortic rings were treated with (<b>A</b>) TMP, (<b>B</b>) crocin, (<b>C</b>) ferulic acid and (<b>D</b>) chlorogenic acid at increasing concentrations. After the treatments, both endothelium-intact (E+) and endothelium-denuded (E−) ring relaxation were measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088243#s2" target="_blank">Methods</a>. Data are mean ± SEM (n = 4–7). ^##<i>P</i><0.01 vs. E+; ^#<i>P</i><0.05 vs. E+.</p

EX-527 abolished TMP-induced PGC-1α up-regulation.

<p>Levels of PGC-1α mRNA were determined by quantitative RT-PCR in the treated bEnd.3 (<b>A</b>) and HUVEC cells (<b>B</b>). Data are mean ± SEM (n = 4). ^#<i>P</i><0.05 vs. Control; ^*<i>P</i><0.05 vs. High glucose; ^$<i>P</i><0.05 vs. High glucose +TMP; ^$$<i>P</i><0.01 vs. High glucose +TMP.</p