Figure 4.

BRD4-driven EZH2 is important for the CSC phenotype A HSC-5 Control and EZH2 CRISPR-depleted cells were seeded in spheroid growth conditions and spheroid number monitored for 9 d. B/C The indicated cell lines were seeded in invasion and migration assays and monitored over time. D The indicated cell lines were grown as spheroids then harvested for immunoblot.E C-MYC plasmid was expressed in BRD4 or EZH2 CRISPR depleted cells and lysates collected for immunoblot of ΔNp63α. F HSC-5 cells (4×10⁴) were plated in spheroid growth conditions and at the time of seeding, 0 or 2 μM GSK126 was added. Incubation continued for 9 d without addition of fresh GSK126 or medium, and spheroid number was counted at each time point G/H HSC-5 spheroid-derived cells were seeded for invasion and migration assays ± GSK126 and monitored over time. I Spheroids were grown for 8 d and then treated with DMSO or GSK126 for 48 h upon which lysates were collected for immunoblots to detect the indicated epitopes. J HSC-5 spheroid-derived cells were injected at 2.5×10⁶ cells per site into nude mice, and mice treated with 0 or 25 mg/kg GSK126. Tumor volume was determined using caliper measurements. The values are mean ± SEM (n=12). Asterisks indicate significant difference in tumor size between control and treated groups. K Protein extracts were prepared from tumors for immunoblotting to detect the indicated epitopes. L Tumors from control and GSK126-treated mice were dissociated to create single-cell suspensions, and tumor cells were seeded to monitor spheroid growth.