We evaluated cell survival by a clonogenic assay; apoptosis by Annexin V immunofluorescence; γH2AX, Rad51, and HDAC4 by immunofluorescence staining; HDAC4, Rad51, and ubiquitin-conjugating enzyme 9 (Ubc9) in HCC cell nuclei by cell fractionation and confocal microscopy; physical interaction between HDAC4/Rad51/Ubc9 by immunoprecipitation; and the downstream targets of HDAC4 knockdown by immunoblotting.
Although Nur77 attenuates sumoylation and stabilizes PEPCK1 via impairing p300 activity and preventing the Ubc9-PEPCK1 interaction, Nur77 is silenced in HCC samples due to Snail-mediated DNA methylation of the Nur77 promoter.
We found that relative to adjacent normal tissues, UBC9 was markedly overexpressed in HCC, which closely correlated with tumor size, tumor microsatellite formation, and tumor encapsulation.