Fig. 4: STAT3 regulates MEF2A-dependent transcription.

A PCMs were transfected with a 4xMEF2 luciferase reporter gene along with two independent siRNAs, siSTAT3#1 and #2, to deplete the endogenous STAT3 levels or a scrambled siRNA was used as 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 (Top panel). Corresponding western blot analysis using equal amounts of total protein of the cell lysates were used to confirm siRNA mediated STAT3 depletion as compared to scrambled controls (Bottom panel). Number of biological replicates for western blotting was n = 3. A schematic of 4xMEF2A-Luc construct is shown below the luciferase results. B PCMs were transfected with an α-MHC-Luc reporter gene with two independent siRNAs, siSTAT3#1 and #2, or scrambled siRNA as control. Luciferase activity was assessed after 48 h and normalized to Renilla activity (Top panel). Corresponding western blot analysis of cell lysate to confirm the depletion of STAT3 protein level as compared to the control is shown (Bottom panel). Number of biological replicates for this analysis was n = 3. Schematic of α-MHC-Luc construct is shown below the luciferase data. C PCMs were transiently transfected with 2xSTAT-Luc reporter gene with two independent siRNAs, siMEF2A#1 and #2, or scrambled siRNA as control. Luciferase activity was assessed after 48 h and normalized to Renilla activity (Top panel). Corresponding western blot analysis of cell lysate to confirm the depletion of MEF2A protein level as compared to the control (Bottom panel). Number of biological replicates carried out for western blotting was n = 3. A schematic of the 2xSTAT-Luc construct used is shown below the luciferase data. D PCMs were transiently transfected with a Flag-HDAC3 construct and lysates were assessed for protein expression by Western blotting. Flag-MEF2A lysate was used for IP using α-Flag magnetic beads and the eluates were blotted with anti-Flag and STAT3 antibodies. IP with lysate from non-transfected PCMs were used as negative controls. Number of biological replicates for western blot and IP carried out was n = 3. Each condition in the luciferase reporter assay is compared to the 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). Dunnett’s multiple comparisons test in one-way analysis of variance using GraphPad Prism 8.0 was used to test for statistical significance. Adjusted p-value *≤0.05, **≤0.01, ***≤0.001****<0.0001, comparing to control.