Journal: Frontiers in Chemistry

Article Title: Activation of GATA4 gene expression at the early stage of cardiac specification

doi: 10.3389/fchem.2014.00012

Figure Lengend Snippet: Valproic acid-enhanced cardiac differentiation. (A) P19 stem cells were treated with DMSO or increasing concentrations of valproic acid (VPA 0.5, 1, 2 mM) during EB formation, maintained in the tissue culture dishes for 3 additional days without treatments, and then stained for myosin heavy chain and cTnT. Quantification is presented as fractions of cells differentiated into cardiomyocytes relative to the total cell populations. Error bars represent the standard deviations of three independent experiments. (B) Shown are the representative microscopy images of the cells stained for cTnT (green). Hoechst was used to stain the DNA (blue) concomitantly (scale bars = 50 μm). (C) Western analysis of GATA4 protein expression and the levels of global H3 acetylation. The blots were then stripped and reprobed for β-tubulin as loading controls. Undifferentiated cells were included as a negative control. Shown are the cropped blot images representing indicated protein. (D) Occupancy of p300 at the GATA4 promoter (GATApro) and a control locus (GATActl) were examined by a real-time PCR based ChIP analysis. Quantification is presented as the fold variations of undifferentiated control.

Article Snippet: The P19 EBs were crosslinked with 1% formaldehyde and lysed using ChIP Lysis Buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1%SDS, 1X protease inhibitors, 1 mM DTT, 1 mM PMSF), and then sonicated with the Bioruptor system (Diagenode).

Techniques: Staining, Microscopy, Western Blot, Expressing, Negative Control, Control, Real-time Polymerase Chain Reaction

Journal: Frontiers in Chemistry

Article Title: Activation of GATA4 gene expression at the early stage of cardiac specification

doi: 10.3389/fchem.2014.00012

Figure Lengend Snippet: Occupancy of p300 at the GATA4 promoter at early stage of differentiation. (A) P19 cells were differentiated with DMSO and co-treatment of curcumin (10 μM) was during the first 2 days of EB formation. The cellular levels of H3 acetylation and p300 protein were analyzed by Western blotting on day 4. The blots were then stripped and reprobed for β-tubulin as loading controls. Undifferentiated cells were used as the negative control. Shown are the cropped blot images representing indicated protein. (B) Quantification of acetylated H3 blots is presented as fold variations of the undifferentiated control (mean ± SD , n = 3). (C) The levels of acetylated H3 at the GATA4 promoter were determined by the ChIP analysis. Quantification is presented as fold variations of the undifferentiated control. (D) Occupancy of p300 at the GATA4 promoter was examined in parallel. (E) Quantification of the p300 Western blots is presented as fold variations of the undifferentiated controls (mean ± SD , n = 3).

Article Snippet: The P19 EBs were crosslinked with 1% formaldehyde and lysed using ChIP Lysis Buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1%SDS, 1X protease inhibitors, 1 mM DTT, 1 mM PMSF), and then sonicated with the Bioruptor system (Diagenode).

Techniques: Western Blot, Negative Control, Control