Effects of homocysteine on MAPKs and apoptotic proteins expression in BMSCs.

<p>(a) The total and phosphorylated JNK, p38 and ERK1/2 protein was detected by western blotting in BMSCs after treatment with homocysteine at the time point of 0, 2, 4, 8, 12 and 24 h. Homocysteine effectively activated phosphorylated JNK expression after treatment with homocysteine. But homocysteine did not increase the expression of total JNK protein in BMSCs. (b) Influences of homocysteine on the expression of Bcl-2, caspase-3, cleaved caspase-3, and p-p53 proteins in BMSCs. n = 3 independent experiments.</p

JNK signal is involved in the apoptosis of BMSCs induced by homocysteine.

<p>JNK specific inhibitor effectively attenuated the apoptosis induced by homocysteine 300 µM in BMSCs. However, p38 and ERK specific inhibitors did not affect homocysteine-induced apoptotic morphological changes in BMSCs.</p

Effects of homocysteine on intracellular ROS and mitochondrial membrane potential of BMSCs.

<p>(a) Intracellular ROS level was measured in BMSCs treated with homocysteine 30, 100 and 300 µM for 24 h by DCFH-DA staining. The ROS level was gradually increased with the increase of homocysteine concentration. (b) Homocysteine induced an obvious depolarization of mitochondrial membrane potential in BMSCs apoptosis by JC-1 staining.</p

Effects of difference concentrations of homocysteine on the morphological appearance and cellular viability of BMSCs.

<p>(a) The morphology of cultured BMSCs was observed after treatment with homocysteine 30, 100, 300 and 1000 µM for 24 h. Homocysteine caused aberrant morphological appearance of BMSCs. (b) Homocysteine significantly decreased the cellular viability of BMSCs in a concentration-dependent manner. * p<0.05 versus Control.</p

Expression of PKCε and HSP60 in miR-1Tg and LNA-antimiR-1 (LNA-1) treated mice before and after ischemia/reperfusion injury.

<p>A, PKCε, protein kinase C epsilon; B, HSP60, heat shock protein 60. Data are expressed as mean±SEM; n = 5; *P<0.05 <i>v</i>s WT, #P<0.05 <i>vs</i> IR.</p

Increased ROS is required for homocysteine-induced apoptosis of BMSCs.

<p>(a) DMTU and NAC attenuated the increase of ROS level by homocysteine in BMSCs. (b) Effects of DMTU and NAC on the apoptotic appearance of BMSCs. The inhibition of ROS with DMTU and NAC abolished the apoptosis of BMSCs induced by homocysteine. (c) Homocysteine-induced depolarization of mitochondrial membrane potential was also effectively reserved by DMTU and NAC in BMSCs.</p

Effects of miR-1 on cardiac infarct area of mice after ischemia/reperfusion injury in mice.

<p>A. Representative images showing infarct areas; B. Statistical analysis of IA/AAR ratio. LNA-1, LNA-antimiR-1; Scramble, scramble locked nucleic acid; IA, infarct area; AAR, area at risk. Data are expressed as mean±SEM; n = 8; *P<0.05 <i>vs</i> WT.</p

Echocardiography of wild type (WT) and miR-1 transgenic (miR-1 Tg) mice.

<p>EF, eject fraction; FS, fractional shortening; LVDd, left ventricle diastolic diameter; LVSd, left ventricle systolic diameter; IVSd, interventricular septum diastolic thickness; IVSs interventricular septum systolic thickness. Data are expressed as mean±SD; n = 6 for each group.</p

Homocysteine induced apoptotic cellular changes of BMSCs.

<p>(a) AO/EB double staining demonstrated the effects of difference concentrations of homocysteine on the apoptosis of BMSCs. BMSCs were incubated with homocysteine for 24 h. (b) Hoeschest33342 staining detected the changes in the nucleus of BMSCs after treatment with homocysteine 30, 100 and 300 µM (scale bar, 20 µm). (c, e) The inhibitory effects of homocysteine on BMSCs were determined by Live/Dead staining. (d, f) TUNEL was used to determine the effects of homocysteine on BMSCs apoptosis (n = 3 independent experiments). * p<0.05 versus Control.</p

Verification of PKCε and HSP60 as targets of miR-1.

<p>A. Sequence alignment between miR-1 and the 3′UTRs of PKCε and HSP60 of human and mouse. B, C. Luciferase reporter activities of chimeric vectors carrying luciferase gene and a fragment of PKCε or HSP60 3′UTR containing the binding sites of miR-1. Data are expressed as mean±SEM; n = 4; *P<0.05 <i>vs</i> control, #P<0.05 <i>vs</i> miR-1.</p