Figure 3.

YB-1 may regulate cardiac function via MEF2B. (A) MEF2B mRNA level in mouse heart tissues detected by real-time qPCR. n = 6 for each group. (B,C) Representative western blots and scatter blots showing MEF2B nuclear protein level in mouse heart tissues from indicated experimental groups. (D,E) Representative immunoblotting and scatter blots demonstrating MEF2B nuclear protein level in H9c2 cells with or without YB-1 overexpression. (B–E) n = 3 for each group. (F) Luciferase activity of pGL3-MEF2B-promoter reporter plasmids stimulated by various YB-1 overexpression degree in H9c2 cells. (G) Luciferase activity of pGL3-MEF2B-promoter reporter in YB-1 knock down H9c2 cells or scrambled shRNA lentivirus infected H9c2 cells, stimulated with HG or not. (H) Luciferase activity of pGL3-MEF2B-promoter reporter plasmids in YB-1 overexpression H9c2 cells or controls, stimulated with HG or not. (I) Luciferase activity of pGL3 (−2,022/+59), pGL3 (−1,393/+59), pGL3 (−919/+59), pGL3 (−440/+59), and pGL3-basic reporter plasmids stimulated by YB-1 overexpression in H9c2 cells. (J) Schematic illustration of mutant of the binding sites of YB-1 on MEF2B promoter in the predicted region of −1,053 to −1,045 (upper part). Luciferase activity was measured in H9c2 cells with mutant or wild type vector stimulated by YB-1 overexpression (lower part). (K,L) ChIP (chromatin immunoprecipitation) result was represented by agarose gel electrophoresis and real time qPCR which revealed the interaction of YB-1 and the promoter region of MEF2B. (F–L) n = 8 for each group. Data were represented as fold of control, mean ± SD. Con, control; DM, diabetes mellitus; DM + PC, diabetes mellitus with PC treatment; shRNAc, scrambled non-specific shRNA; shRNA YB-1, YB-1 specific shRNA; NG, 5.5 mmol/L D(+) glucose; HG, 25 mmol/L D (+) glucose; OE, overexpression; WT, wild type; Mut, Mutant. *P < 0.05, **P < 0.01. Student's t-test or two-way ANOVA, Bonferroni comparison test.