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Fig. 1 | Clinical Epigenetics

Fig. 1

From: Differential effect of histone H3.3 depletion on retroviral repression in embryonic stem cells

Fig. 1

Measuring of H3.3 deposition and dynamics on the MLV provirus. A Illustrating the retroviral insert with the PBSpro and PBSgln variation, the primers used are marked by pink or blue lines, respectively. B Anti-HA ChIP on MLV-infected, Dox-induced KH2 cells followed by RT-qPCR on proviral and genomic targets. Values are % Input normalized to the b-Globin gene. Data are the mean ± s.e.m. (n = 4). C A schematic diagram showing the doxycycline (Dox) histone H3.3-HA induction system and the use of this system for assaying H3.3 turnover dynamics. HA-tagged H3.3 expression was induced by Dox addition at different times and immunolabeled with anti-HA antibodies. DAPI staining overlay is shown in blue (D) immunoblotting of Dox-induced KH2 cells to extract for the indicated hours using anti-HA, with anti-H3 and GAPDH as loading controls. E Accumulation of H3.3-HA into PBSpro, F PBSgln and G genomic sequences at 1, 4 and 8 h after DOX induction. Presented are Bound values normalized to the 8 h Bound fraction. ChIP-qPCR data are the mean ± s.e.m. (n = 3 for PBSpro and PBSgln, n = 4 for the genomic sequences). H Turnover rate of the proviral and genomic sequences was measured using log2 of %Input ChIP data for 1H divided by 8H. Data are the mean ± s.e.m. (n = 3–4). All P values were calculated using two-tailed unpaired Mann–Whitney U test, *P < 0.05, ***P < 0.001, ****P < 0.0001

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