Journal: Cell Death & Disease

Article Title: The MEF2A transcription factor interactome in cardiomyocytes

doi: 10.1038/s41419-023-05665-8

Figure Lengend Snippet: A PCMs were transfected with a Flag-MEF2A construct and lysates were assessed for expression by Western blotting. Flag-MEF2A lysates were used for IP using α-Flag magnetic beads and the eluates were blotted with anti-Flag and STAT3 antibodies. IP with lysates from non-transfected PCMs were used as negative controls (Left panel). Non-transfected PCM lysates were used for IP using MEF2A antibody to confirm the interaction between endogenous MEF2A and RBPMS. IP with IgG was used as controls (Top right). Non-transfected PCM lysates were used for IP using a STAT3 antibody IP to confirm the interaction between endogenous STAT3 and MEF2A. IP with IgG was used as control (Bottom right). Number of biological replicates carried out for the Western blot and IP data is n = 3. B Confocal immunofluorescence analysis of endogenous MEF2A and STAT3 indicates a nuclear localization in PCMs. PCMs were fixed and stained for MEF2A in red and counterstained for STAT3 in green. Slides were analyzed by confocal microscopy. The scale bars are 2 and 10 μm. The intensity blot of MEF2A and STAT3 signals is shown over the region shown by the yellow line in the merged image. C STAT3 and MEF2A expressing vectors alone and in combination were ectopically expressed in PCMs along with a 4xMEF2 luciferase reporter gene. 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. D PCMs were transiently transfected with 4xMEF2 luciferase reporter gene for 48 h and then treated with STAT3 inhibitor (C188-9;10 µM) for 1 h in serum free medium. The control cells were treated by the solvent (DMSO) in serum free medium. Representative western blots of three independent biological replicates are presented which were carried out to assess the STAT3 inhibitor effect as compared to control condition. A schematic of the 4xMEF2A-Luc construct is shown below the luciferase data. Each condition in the luciferase reporter assay 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 and independent two sample t -test 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.

Article Snippet: Tukey’s Multiple comparisons test in one-way ANOVA using GraphPad Prism 8.0 was used to test for statistical significance.

Techniques: Transfection, Construct, Expressing, Western Blot, Magnetic Beads, Immunofluorescence, Staining, Confocal Microscopy, Luciferase, Activity Assay, Reporter Assay

Journal: Cell Death & Disease

Article Title: The MEF2A transcription factor interactome in cardiomyocytes

doi: 10.1038/s41419-023-05665-8

Figure Lengend Snippet: 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.

Article Snippet: Tukey’s Multiple comparisons test in one-way ANOVA using GraphPad Prism 8.0 was used to test for statistical significance.

Techniques: Transfection, Luciferase, Activity Assay, Western Blot, Construct, Expressing, Magnetic Beads, Reporter Assay

Journal: Cell Death & Disease

Article Title: The MEF2A transcription factor interactome in cardiomyocytes

doi: 10.1038/s41419-023-05665-8

Figure Lengend Snippet: 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.

Article Snippet: Tukey’s Multiple comparisons test in one-way ANOVA using GraphPad Prism 8.0 was used to test for statistical significance.

Techniques: Transfection, Luciferase, Activity Assay, Western Blot, Construct, Expressing

Journal: Cell Death & Disease

Article Title: The MEF2A transcription factor interactome in cardiomyocytes

doi: 10.1038/s41419-023-05665-8

Figure Lengend Snippet: A MEF2A and STAT3 are involved in the increase of cardiomyocyte surface area in response to phenylephrine. Immunofluorescence analysis of α-Actinin and DAPI was performed in PCMs transfected with siRNA targeting MEF2A and STAT3 individually and in combination for 48 h then treated with PE (200 μM) or vehicle (H 2 O) for a further 48 h. The figure illustrates 2 representative images for each condition. The scale bar is 20 μm. B The scatter plot represents the quantification of the surface area of α-Actinin-positive cells using Image J software. The blue dots represent PCMs treated with H 2 O and purple dots represent the PCMs treated with PE. The data presented are derived from the surface areas of 100 cells that were randomly selected from four biological replicates (25 cells per biological replicate). Each cell surface area measurement is represented as a dot in each condition and the mean is indicated by a horizontal line. Tukey’s Multiple comparisons test in one-way ANOVA using GraphPad Prism 8.0 was used to test for statistical significance. * represents statistical significance from control vehicle-treated group. # represents statistical significance from PE-treated group. Adjusted p -value *≤0.05, **≤0.01, ***≤0.001, ****<0.0001, comparing to control.

Article Snippet: Tukey’s Multiple comparisons test in one-way ANOVA using GraphPad Prism 8.0 was used to test for statistical significance.

Techniques: Immunofluorescence, Transfection, Software, Derivative Assay