Effects of miR-20b on the BMP signaling pathway, as assessed by by western blotting.

<p>a): in the miR-20b overexpression group; b): in miR-20b silenced group. (n = 3, *: P<0.05, **: P<0.01, ***: P<0.001)</p

The relative expression of miR-20b in ventricular septal defect.

<p>VSD: ventricular septal defect group; control: healthy group.</p

The expression of p-ERK1/2, p-JNK, Bcl-2 and Bax.

<p>The effects of luteolin and SP600125 on the expression of total ERK and p-ERK (A, B), Bcl-2 (C, D) total JNK and p-JNK (E, F), Bax (G, H). After 2 h reperfusion, the myocytes were harvested to detect protein expressions by western blot analysis. The results were expressed as the mean ± SEM. n = 3. ^**<i>P</i><0.01 versus DMSO; ^#<i>P</i><0.05, ^##<i>P</i><0.01 versus I/R; ^$<i>P</i><0.05 versus I/R+Lut (8.0 µM), ^&<i>P</i><0.05, ^&&<i>P</i><0.01 versus I/R+Lut(8.0 µM)+PD(10 µM).</p

Effects of SAA on MIA and LDH of coronary effluent.

<p>(<b>a</b>) Effects of SAA and SP600125 on MIA. After the ventricular tissue reperfusion was finished, the tissue was sliced into 1-mm sections. The weight of the infarction tissue was expressed as a percentage of the total ventricle weight. (<b>b</b>) Effects of SAA and SP600125 on LDH of coronary effluent. After 15-min reperfusion, the coronary effluent of each group was collected for LDH assay. *P<0.05, **P<0.01 versus CON group, ^$P<0.05, ^$$P<0.01 versus I/R, ^#P<0.05, ^##P<0.01 versus SAA+I/R, ^&P<0.05, ^&&P<0.01 versus PD+SAA+I/R. All data were expressed as mean ±SEM, n = 6.</p

Luciferase activity assessed by the Dual Luciferase Reporter Assay System.

<p><b>a) Bambi confirmed as the direct target gene of miR-20b.</b> 20b+Bambi: the luciferase activity of Bambi in the miR-20b overexpression group; NC+Bambi: the luciferase activity of Bambi in the control; 20b+mut: the luciferase activity of mutated Bambi in the miR-20b overexpression group. <b>b) miR-20b silencing confirmed. c) miR-20b silencing constant during the differentiation (day 10)</b> (n = 6, ***: P<0.001)</p

Effects of miR-20b on cell apoptosis.

<p>a) Apoptosis assayed by binding to Annexin V-APC/7-AAD. b) Apoptosis detected by measurement of Caspase-3 activity. miR-20b-over: miR-20b overexpression cells; miR-20b-in: miR-20b silenced cells. (n = 4, *: P<0.05, **: P<0.01)</p

Effects of miR-20b on mitochondrial membrane potential (MMP) in differentiated cells.

<p>A: control for miR-20b overexpression; B: miR-20b overexpression; C: control for miR-20b silencing; D: miR-20b silencing. (*: P<0.05)</p

The morphological changes during P19 differentiation (×10) b) The expression of the cTnT protein during differentiation (a).

<p>To investigate the differentiation of P19 cells into mature cardiomyocytes, we used western blotting to identify the expression of the cTnT protein during differentiation. The expression of β-actin was used as a internal control.</p

Effects of miR-20b on the mitochondrial DNA (mtDNA) copy number.

<p>On the 10th day of differentiation, cellular mtDNA content was assessed by qRT-PCR analysis with primers designed to target the <i>CYTB</i> and 28 S rRNA genes (n = 6). miR-20b-over: miR-20b overexpression cells; miR-20b-in: miR-20b silenced cells. P > 0.05 in comparison with negative control (NC) cells.</p

The expression of p-PP1a, p-PLB and SERCA2a.

<p>The effects of luteolin and SP600125 on the expression of total PP1a and phospho-PP1a (A, B), total PLB and phospho-PLB (C, D), SERCA (E, F). The results were expressed as the mean ± SEM, n = 3. ^*<i>P</i><0.05,^**<i>P</i><0.01 versus DMSO; ^#<i>P</i><0.05 versus I/R; ^$<i>P</i><0.05 versus I/R+Lut (8.0 µM); ^&<i>P</i><0.05 versus I/R+Lut(8.0 µM)+PD(10 µM).</p