Journal: Nucleic Acids Research
Article Title: Molecular basis for SOX2-dependent regulation of super-enhancer activity
doi: 10.1093/nar/gkad908
Figure Lengend Snippet: Confocal microscopic analysis of enhancer activity of mSE078 fragments using PCP-PP7 system in MEF cells. ( A ) Left: Schematic representation of mkrn1 P -12 × PP7-SE reporter plasmid construct. Right: Schematic representation of PCP-PP7 system used in this study. ( B ) Confocal microscopic analysis of tdPCP-CFP (transcript), SOX2-mCherry and p300-GFP signals in MEF cells. Cells were imaged after 24 h-post transfections of four plasmids. Left: 40× magnification images; Right: closed-up images of single cells. ( C ) Quantification of CFP (PCP), mCherry (SOX2) and GFP (p300) foci. Error bars indicate standard errors of means (SEM) from at least 15 nuclei. Quantification was done using ImageJ software. Particle size larger than 2 × 2 μm 2 was calculated. ( D ) Pearson's coefficients of CFP and GFP signals to mCherry signals. Pearson's coefficients were calculated using ImageJ software. Error bars indicate SEMs from ten nuclei. ( E ) Fluorescence recovery after photobleaching (FRAP) measuring SOX2-mCherry condensates in MEF cells co-transfected with PCP-CFP, SOX2-mCherry and p300-GFP plasmids. Photobleaching was initiated at 0 s. Curve shows mean (red dot) and SEM (black bar) of mCherry intensity of five regions. FRAP recovery curves were fitted to the double exponential function. P -values were calculated using one-way ANOVA. (ns) P < 0.1234; (*) P < 0.0332; (**) P < 0.0021; (***) P < 0.0002; (****) P < 0.0001.
Article Snippet: FRAP recovery curves were fitted to the double exponential function in GraphPad Prism software v.8.3.0 (GraphPad software, Boston, MA, USA).
Techniques: Activity Assay, Plasmid Preparation, Construct, Transfection, Software, Fluorescence