Figure 4

Generation of MYC enhancer region-deleted Huh7 cells via the CRISPR/Cas9 system. (A) Schematic illustration of the MYC enhancer locus structure and the wild-type (WT) and mutant (ΔR3) allele sequences around the R3 target region of Huh7 cells. (B) qRT-PCR results showing the levels of MYC mRNA, (C) R2 and R3-associated eRNAs in WT and R3-edited Huh7 cells. (D) TAD of MYC from Hi-C matrix of heatmap on chr8:126,840,000–130,080,000 in HepG2 cells (http://3dgenome.fsm.northwestern.edu/view.php). (E) qRT-PCR results showing the levels of MYC-related lncRNA and (F) MYC-related gene mRNAs in WT and R3-edited Huh7 cells. 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. WT and edited Huh7 cells were cultured in a growth medium for 96 h. The obtained absorbance was normalized to each 0 h absorbance. The data represent three biologically independent experiments. **p < 0.01. (H) Colony formation ability of WT and R3-edited Huh7 cells. Cells were grown for 10 days and stained with Crystal Violet. The relative colony formation efficiency was measured as a percentage of the area covered by the colonies. The data represent three biologically independent experiments. **p < 0.01. (I) WT and R3-edited Huh7 cells were cultured in cancer stem cell (CSC) growth media for a spheroid formation assay under ultralow adherence conditions. Cells grown for 7 days and 14 days in spheroid-forming conditions are shown in bright-field images taken with a 4X 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.