Fig. 7: MEF2A/STAT3 complex regulates the MMP9 expression in cardiomyocytes.

A PCMs were transiently transfected with an MMP9 luciferase reporter gene along with siRNA targeting the endogenous MEF2A and STAT3 levels individually and in combination. Scrambled siRNA was used as a control. Renilla luciferase was used to normalize for transfection efficiency. Lysates were collected at 48 h. The luciferase activity under each condition was measured and normalized to Renilla values to determine the fold change. N = 3 biological replicates per condition (Top panel). A representative western blot for three biological replicates for MEF2A, STAT3, MMP9, and β-actin is presented. Corresponding western blot analysis was used to confirm siRNA mediated MEF2A and STAT3 depletion as compared to scrambled controls (Bottom panel). A schematic of the MMP9-Luc construct is shown below the representative western blot. The expression of MEF2A, STAT3, and MMP9 are assessed using western blot analysis. The dot graphs represent the level of MEF2A, STAT3, and MMP9 proteins after normalization to β-actin (right panel). Both prominent bands (hyper and hypophosphrylated) for MEF2A are included together in the quantification. Data are presented as mean ± SEM. n = 3 *P < 0.05 vs control. B PCMs were transfected with the MMP9 luciferase reporter gene and harvested at 48 h. On day1 after transfection, cells were treated with the p38 MAPK inhibitor (SB203580; 10 μM) or its inactive analogue (SB202474; 10 μM) for 24 h in a serum-free medium. Cells were treated with a STAT3 inhibitor (C188-9;10 µM) as indicated, and the control cells were treated with the corresponding solvent (DMSO). Luciferase values were normalized to Renilla. N = 3 biological replicates per condition. Representative western blot for three biological replicates for MEF2A, pSTAT3, STAT3, MMP9, and β-actin. Corresponding western blot analysis of the cell lysates was used to confirm SB203580 and C188-9 treatments as compared to controls as indicated (Bottom Panel). C The plot represents the MMP9 protein levels after normalization to β-actin. Each condition in the luciferase reporter is compared to the respective control condition for three independently transfected samples (technical replicates) to determine the fold change. Each dot represents one biological replicate and corresponds to the mean of three technical replicates. The error bars represent standard error of the mean (SEM). Tukey’s multiple comparisons test in one-way ANOVA using GraphPad Prism 8.0 were used to test for statistical significance. Adjusted p-value *≤0.05, **≤0.01, ***≤0.001, ****<0.0001, comparing to control.