Figure 5.

EZH2 interacts with STAT3 to regulate the CSC phenotype A Control and EZH2-sgRNA expressing cells were harvested for immunoblot and probed for the indicated epitopes. B HSC-5 spheroids were treated with GSK126 for 48 h then lysates collected for immunoblotting. C HSC-5 spheroids were harvested for protein extraction for immunoprecipitation/immunoblot. D HSC-5 spheroids were treated with GSK126 or DMSO for 48 h prior to lysates being collected for immunoprecipitation and immunoblot. HSC-5 cells expressing EV or STAT3C were treated with GSK126 and spheroid formation (E) and invasion (F) were monitored. G Protein lysates were collected for western blot of the indicated proteins. H STAT3 CRISPR depleted HSC-5 cells containing wildtype STAT3 or methylation mutant K180A STAT3 were grown as spheroids for 10 days and spheroid number counted. I STAT3 CRISPR depleted HSC-5 cells containing wildtype STAT3 or methylation mutant K180A STAT3 were seeded on a Matrigel coated membrane for invasion assays. J Protein lysates were collected from STAT3 CRISPR depleted HSC-5 cells containing wildtype STAT3 or methylation mutant K180A STAT3 and immunoblots run for the indicated epitopes. K Protein lysates were collected from STAT3 CRISPR depleted HSC-5 cells containing wildtype STAT3 or methylation mutant K180A STAT3 and Co-Immunoprecipitation done for the indicated proteins. L HSC-5 spheroid-derived cells were injected as described above and mice were treated with 0 or 25 mg/kg STATTIC. Tumor volume caliper measurements are mean ± SEM (n=8). Asterisks indicate significant difference in volume between control and treated groups Representative Control and STATTIC treated tumors were harvested on week seven and photographed. M Protein extracts were prepared from tumors for western blot to detect the indicated epitopes. N Tumors from Control and STATTIC treated mice were used to create single-cell suspensions, and cells were seeded in non-attached conditions to monitor the ability to form spheroids.