Figure 5.

Function of MLL1 in regulation of H3K4me3 in the CD274 promoter and PD-L1 expression in human pancreatic cancer cells. A) Chaetocin is a potent MLL1 inhibitor. Chaetocin was tested in a 10-dose IC₅₀ mode with threefold serial dilutions using ³H-S-adenosyl-methionine as substrate with MLL1 recombinant protein complex. The enzyme activity was then analyzed and plotted against chaetocin concentrations. IC₅₀ was calculated using the Graphpad Prism program. B) PANC10.05 cells were treated with chaetocin (50 nM) for two days and analyzed with H3K4me3-specific monoclonal antibody (MAb) by chromatin immunoprecipitation (ChIP). The ChIP DNA was amplified using ChIP primer 4. The relative H3K4me3 level was normalized to input DNA and analyzed by two-sided t test. C) PANC10.05 cells were treated with chaetocin at the indicated doses for two days, stained with PD-L1-specific MAb, and analyzed by flow cytometry. The PD-L1 protein mean fluorescent intensity is presented at the right panel. Column: mean; bar: SD. D) H3K9me3 is not enriched in the CD274 promoter region in human pancreatic cancer cells. PANC10.05 cells were analyzed by ChIP with H3K9me3-specific MAb and polymerase chain reaction primers. All statistical t tests were two-sided. ChIP = chromatin immunoprecipitation; mAb = monoclonal antibody.