Fig. 7.

VCP increases the protein stability of HMGB1. A–C Western blot was performed to evaluate the half-life of HMGB1 protein in HCC cells. All groups were treated with cycloheximide (CHX, 50 μg/mL), a classic protein synthesis inhibitor, from 0 to 24 h. D–F The GSEA analysis indicated the enriched pathways in HCC patients of high VCP and HMGB1 expressing groups from TCGA database, respectively. The median value of VCP and HMGB1 transcriptional levels in the HCC cohort was regarded as the cut-off. G The total cellular levels of ubiquitylated proteins in HCC cells was determined by western blot. Both Huh7 and MHCC-LM3 cells were treated with MG132 (20 μM, 6 h), which is a general proteasome inhibitor. H The ubiquitylated HMGB1 protein was detected by western blot after HMGB1 immunoprecipitation in HCC cells. Cells were treated with MG132 (20 μM, 6 h) or the VCP inhibitor NMS873 (10 μM, 6 h). I, J The Huh7 cells with VCP depletion and MHCC-LM3 cells with ectopic VCP overexpression were incubated with MG132 (20 μM) for 24 h. The whole-cell lysates were collected and separated by SDS-PAGE. Proteins were detected by indicated antibodies. All *P < 0.05, **P < 0.01, and ****P < 0.0001