Figure 4. Elevated REST expression increases G9a-dependent histone methylation and target gene repression.

A. Western blotting analysis was performed with extracts from isogenic DAOY and DAOY-REST cells to determine the effect of REST elevation on global histone H3K9-me1, -me2, and -me3. Total histone H3 served as the loading control. B. Changes in G9a and REST-target gene expression were evaluated by Q-RT-PCR. C. ChIP assay were performed to demonstrate an increase in histone H3K9 -me1 and -me3 at USP37 promoter region-1 and -me1 at region-2 in DAOY-REST cells relative to DAOY cells. D. MTT assays were used to examine the effect of REST elevation in DAOY-REST cells on its response to G9a inhibition compared to DAOY cells. Solid black line indicates the IC₅₀ value, which is also tabulated. (*=p<0.05, **=p<0.01, ****=p<0.0001, and n.s. = not significant). E. Live-cell nuclear staining assay was performed using DRAQ5 to distinguish between cytotoxic and cytostatic effects of UNC 0638 treatment on DAOY and DAOY-REST cells. F. Western blotting analysis was performed to measure the levels of the GLP target protein c-Myc following treatment of DAOY and DAOY-REST cells with 48hrs-IC₅₀ doses of UNC 0638. β-actin was used as a loading control.