Gsk126

In order to interfere with the production of H3K27me3 in pGCs, GSK-126 (#1346574-57-9, MedChemExpress, Monmouth Junction, NJ, USA) and GSK-J4 (#1373423-53-0, MedChemExpress, Monmouth Junction, NJ, USA) were chosen to be the antagonist and agonist of H3K27me3, respectively [29 (link)]. The doses of antagonist GSK-126 and agonist GSK-J4 were 6 and 2 nM. The negative controls of antagonist GSK-126 and agonist GSK-J4 were 6 and 2 nM of a dimethyl sulfoxide (DMSO) solvent that was used to dissolve the antagonist and agonist. The effect of the drugs was measured by WB. The performances of WB were the same as above. The antibodies of H3K27me3 were purchased from Millipore (#07-449, Millipore, Germany). GAPDH (Sigma, St. Louis, MO, USA) was used as reference protein. Each group featured three replications for cell treatments and two replications for WB.

DHT (4,5α-Dihydrotestosterone) was purchased from Sigma-Aldrich (St. Louis, MO, USA; A8380) and dissolved in ethanol at 10 μM to be used at a final concentration of 10 nM. MDV3100 (enzalutamide), from Santa Cruz Biotechnology (Dallas, TX, USA; sc-364354), was diluted in DMSO at 10 mM and used at 10µM. GSK126 (EZH2 inhibitor) was purchased from MedChemExpress (Monmouth Junction, NJ, USA; HY-13470), dissolved in DMSO, and used in a range of concentrations for 24 h. C2 Ceramide (N-acetoyl-D-erythro-sphingosine) was purchased from Avanti Polar Lipids (Alabaster, AL, US; 860502), dissolved in DMSO, and used at a final concentration of 20 μM. Doxycycline Hyclate (Dox, used at a final concentration of 0.25 μg/mL) and C16 Ceramide 1-phosphate (C1P, 860533P) were purchased from Sigma Aldrich (St. Louis, MO, US). An aqueous dispersion (in the form of liposomes) was prepared by sonicating C1P (1 mg) in sterile nanopure water (600 μL) on ice using a probe sonicator for six cycles of 8 s on and 5 s off until a clear dispersion was obtained. C1P concentration in the stock solution was 2.6 mM and a final concentration of 20 μM was used for all cellular assays. This procedure is considered preferable to C1P dispersions in organic solvents because lipid droplet formation is minimized and exposure of cells to alcohols or dodecane is avoided.

The human hepatoma cell lines PLC/PRF/5, Huh7, and Hep3B used in this study were purchased from the American Type Culture Collection (Manassas, VA, USA). PLC/PRF/5 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium, and Huh7 and Hep3B cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C and 5% CO₂. Hepatoma cells were treated with recombinant interferon gamma (IFNγ) (Sino Biological Inc.), DZNep (MedChemExpress, Monmouth Junction, NJ, USA), or GSK-126 (MedChemExpress) for different times and at different concentrations.
Monocytes were selected from peripheral blood mononuclear cells using anti-CD14 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) as described previously [33 (link)].