CBX7 inhibitors change the epigenetic landscape of OCI-AML3 cells to an active state. (A-B) H2Aub expression levels in OCI-AML3 cells after 4 days of in vitro treatment. (A) Representative histograms of the mean fluorescent intensity (MFI) of H2Aub-AF647 signal obtained with flow cytometry in untreated DMSO control cells (black), and after treatment with increasing concentrations of MS452 (red), EC-134 (purple), BDA-41 (blue), or UNC0642 (orange); see supplemental Figure 2A for complete gating strategy. (B) Quantification of the MFI of H2Aub-AF647 normalized to the DMSO control. Bars represent the mean of ≥3 replicates with SEM as error bar. Student t test was used to calculate the P value between DMSO and treatment, ∗P < .05. (C-D) CBX7 binding to the chromatin at (C) cell cycle arrest and (D) differentiation-associated target genes after 24 hours of treatment with MS452, EC-134, or BDA-41 in OCI-AML3 cells overexpressing the fusion protein CBX7-GFP. Chromatin immunoprecipitation quantitative PCR (qPCR) experiments were performed to calculate percent input, which was used to normalize the CBX7 binding in the treated samples to their experimental DMSO control; see supplemental Table 1 for raw data. Bars represent the mean of ≥1 replicates with SEM as error bar. Student t test was used to calculate the P value between DMSO and treatment, ∗P < .05. (E-F) Upregulation of the (E) cell cycle arrest and (F) differentiation-associated target genes in OCI-AML3 cells after 4 days of treatment with MS452, EC-134, or BDA-41. CDKN1A, INK4A, ID2, IRF4, MAFB, and CD14 relative to HPRT messenger RNA (mRNA) expression were obtained with qPCR and normalized to their experimental DMSO control (2^−ΔΔCt). Bars represent the mean of ≥2 replicates with SEM as error bar. Student t test was used to calculate the P value between DMSO and treatment, ∗P < .05.