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Fig. 6 | Epigenetics & Chromatin

Fig. 6

From: DNA replication dynamics of vole genome and its epigenetic regulation

Fig. 6

Induced hyperacetylation leads to a decrease in nucleotide incorporation rate and a slower fork speed. a Schematic representation of the calculation of relative nucleotide incorporation rate. Modified thymidine analog EdU was added for 10 min to Microtus cabrerae cells that were treated or untreated with HDACi prior to fixation. EdU was detected with ClickIT chemistry and endogenous PCNA via antibody detection. Whereas EdU represents the amount of synthesized DNA (incorporated nucleotides), PCNA reflects the replication machinery and thus active forks. For the estimation of nucleotide incorporation rate, the ratio of EdU (incorporated nucleotides) and PCNA (replication machinery) was estimated, as a marker for the speed of replication forks. If the ratio shows a value = 1, this means a complete overlap of both signals (EdU and PCNA) and indicates a slow replication fork speed and thus slower replication forks. If the ratio of both signals is > 1, this means that there was more DNA synthesized, indicating faster replication forks and hence a faster fork speed. b The ratio of EdU and PCNA signals was plotted as box plots. Cells were categorized by visual inspection of the EdU signal into early (light gray box) and mid + late cells (dark gray box). HDACi-treated samples are indicated by the red-framed box. In untreated cells, the nucleotide incorporation rate increases over time, demonstrating an increase in fork speed from early to mid + late cells. In contrast to untreated cells, HDACi-treated samples show a significantly lower ratio of EdU/PCNA signal, indicating a slower nucleotide incorporation rate and thus a slower fork speed. Statistical significance was tested using the Wilcoxon test, comparing untreated and HDACi-treated Microtus cabrerae cells. ***P < 0.001

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