Figure 5

Effects of ASOs targeting MYC eRNAs in Huh7 cells. (A) Schematic illustration of the MYC enhancer locus structure and USCS genome browser view of the GRO-seq peak, H3K27ac enrichment, and p300 binding sites along the R2 and R3 regions. The targeting ASOs, ASO R2-P1 and ASO R3-P2, were designed to bind to the R2-S and R3-S eRNAs, respectively. (B,C) Huh7 cells were treated with 125 pmol of a non-targeting ASO (ASO NC) or the targeting ASO. Relative quantitation of R2-S (B) and R3-S (C) eRNA expression levels was performed by qRT-PCR. U6 stands for the control gene RNU6-1. (D–F) Relative quantitation of MYC mRNA expression levels (D) and MYC-controlled mRNA expression levels (E,F). The values are the mean ± SD from triplicate well measurements. *p < 0.05 and **p < 0.01. (G) Cell proliferation was determined using a WST-1 assay and represented by the relative absorbance at 450 nm. Absorbance was measured every 24 h from 0 to 96 h after transfection of ASO. The data represent three biologically independent experiments. **p < 0.01. (H) ASO-transfected Huh7 cells were cultured in a growth medium for the spheroid formation assay. Cells grown for 7 days and 14 days in spheroid-forming conditions are shown in bright-field images taken with a 10X objective. The number of large spheres (over 100 µm) was counted; scale bar = 100 µm. The data represent three biologically independent experiments. **p < 0.01.