Figure 6.

High mobility group box 1 (HMGB1) facilitates enhancer of Zeste homolog 2 (EZH2) methyltransferase activity via protein–protein interaction. A: Cholangiocarcinoma (CCA) cell extracts were subjected to immunoprecipitation (IP) using anti-EZH2 antibody or isotype IgG followed by Western blot analysis. B: Mouse CCA cells were transfected with HMGB1-targeting siRNA or scramble control. Seventy-two hours posttransfection, nuclear extracts were collected and subjected to EZH2 methyltransferase activity assays using H3K27 as substrates. C: Detection of EZH2 and HMGB1 by immunoblotting in mouse CCA cells with EZH2 deletion (by Ad-Cre virus infection) (upper panel) or with HMGB1 knockdown (by siRNA transfection) (lower panel). D: HMGB1 expression levels in human CCAs and non-tumorous tissues of Gene Expression Omnibus data sets GSE76297 and GSE107943. E: HMGB1 levels in CCAs from wild-type mouse at 5 weeks post–Notch1 intercellular domain (NICD)/AKT/Sleeping Beauty (SB) plasmids injection. Liver tissues from noninjected mice were used as controls. F: Correlation of EZH2 and HMGB1 mRNA levels in patient tissues from data sets GSE76297 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE76297) or GSE107943 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE107943). G: Schematic illustration of the mechanisms of EZH2 in CCA. ∗P < 0.05.