MOESM5 of DNA replication dynamics of vole genome and its epigenetic regulation

Additional file 6. Titration analysis of potential HDAC inhibitors. (A) Overview of different HDAC classes and corresponding HDAC inhibitors of each class. MS-275 only affects HDAC1, HDAC2 and HDAC3 of class I (orange), whereas TSA and LBH-589 inhibit HDACs of class I, II and IV (blue/red). (B) Titration analysis of histone hyperacetylation in response to different HDAC inhibitors in female Microtus cabrerae cells. TSA treatment was performed over three days (72 hours). Previous studies have shown that a concentration of 20 nM was sufficient to hyperacetylate very condensed constitutive heterochromatin in C2C12 mouse cells [9]. Here, we increased this concentration 5x, 10x and 20x and did not achieve significant hyperacetylation at heterochromatic sex chromatin of Microtus cabrerae cells. Over time and at higher concentrations of TSA, cells showed morphological changes and with 800 nM cells died. In contrast, treatment with LBH-589 leads to hyperacetylation at sex chromatin already after one day of treatment. We were able to achieve significant increase in histone acetylation levels at the sex chromatin. To ensure a sufficient and stable hyperacetylation in the absence of morphological changes, we treated the cells with 50 nM for one day. MS-275 data are not shown as cells either showed same levels as controls or died directly after treatment. Statistical significance was tested using the t-test, comparing untreated and HDACi-treated cells. Error bars demonstrate 95Cl. ***P < 0.001. (C) Effect of HDACi treatment on histone acetylation level of female Microtus cabrerae cells. Treatment with LBH-589 leads to significantly increased H3K9ac and H4K8 mean acetylation level at sex chromatin. Mean H4K8ac level at untreated samples is significantly higher at the X* in comparison with the X. Statistical significance was tested using the t-test, comparing HDACi-treated cells and untreated cells, as well as mean H4K8ac levels in untreated cells at X* versus X. Error bars demonstrate 95Cl. ***P < 0.001.