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Fig. 4 | Epigenetics & Chromatin

Fig. 4

From: A high-throughput screen of inactive X chromosome reactivation identifies the enhancement of DNA demethylation by 5-aza-2′-dC upon inhibition of ribonucleotide reductase

Fig. 4

Hydroxyurea and 5-aza-2′-dC treatment of myeloid leukemia cell lines. a Dose–response curves measuring viable K562 cells using the Cell-Titer Glow Assay. Chemical treatments were performed with HU or 5-aza-2′-dC (Aza) alone or in combination at a fixed concentration ratio of 4000:1 HU:Aza (based on individual IC50 values). Red lines indicate concentrations assayed in the subsequent RRBS analysis displayed in c. The accompanying table depicts the Chow-Talalay analysis of the Combination Index (CI) at the given treatment combination concentrations. CI values <1.0 indicates synergy, 1= additive effect, and >1.0 antagonism of the combination drug effect. b As in a except dose–response curves for HL60 cells using a fixed concentration ratio of 150:1 HU:Aza in the combined treatments. c Heat map showing an unsupervised hierarchical clustering of X chromosome CpG methylation levels in K562 cells treated with the indicated chemicals for 72 h. Only those CpGs with greater than 0.75 methylation level in the DMSO-treated samples are displayed, as these are the CpGs most dramatically affected by 5-aza-2′-dC treatment (see Fig. 3). CpGs were filtered for at least 10× sequencing coverage across all samples. DNA methylation levels were profiled for the treatments with the HU/aza concentration combinations indicated with red lines in a (labeled Low, Mid, High). In addition, we treated cells with either 5-aza-2′-dC or HU at the respective concentrations (Low HU (0.16 mM), Mid HU (0.4 mM), High HU (0.80 mM), and Low Aza (0.04 μM), Mid Aza (0.1 μM), High Aza (0.2 μM)). Aza was resuspended in DMSO, thus DMSO only controls are matched to volume of Aza added in corresponding Aza samples. d Flow cytometry analysis of propidium iodide-stained K562 cells treated with low and high HU concentrations as described in c compared to low or high DMSO control treatment

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