Recombinant human IFN-γ (#285-IF) IFN-α (#11100-1) and IFN-β (#8499-IF) proteins were purchased from R&D Systems (Minneapolis, MN) and reconstituted in sterile, deionized water. MRT67307 and Ruxolitinib were synthesized and purchased from Shanghai Haoyuan Chemexpress Co. Both drugs were reconstituted at 10 mM in DMSO and stored at −20ºC. GSK126 (#S7061) was purchased from Selleck chemicals (Houston, TX) and reconstituted at 5 mM in DMSO and stored at −20ºC.
For IFN pulse experiments, cells were pulsed 10 minutes with IFN–γ (200 ng/mL or 10 ng/mL), IFN-α (10 000 U/mL) or IFN-β (10 000 U/mL), extensively washed, and chased in fresh media for an additional 24, 48 or 72 hours. To test drug effects on gene expression or protein secretion, IFN–γ pulsed H69AR cells were treated with DMSO, 1μM MRT67307 or 100 nM Ruxolitinib for 24, 48 and 72 hours.
For EZH2 inhibition experiments, H69 cells were treated with 5 μM GSK126 for 6 days. Drug was replenished every 3 days with both suspension and adherent cells carried each time. After the GSK126 treatment period, equal numbers of DMSO-treated and GSK126-treated cells were exposed to either H2O or 200 ng/mL IFN-γ for 24 hours before harvesting of RNA or conditioned media (CM).
For IFN pulse experiments, cells were pulsed 10 minutes with IFN–γ (200 ng/mL or 10 ng/mL), IFN-α (10 000 U/mL) or IFN-β (10 000 U/mL), extensively washed, and chased in fresh media for an additional 24, 48 or 72 hours. To test drug effects on gene expression or protein secretion, IFN–γ pulsed H69AR cells were treated with DMSO, 1μM MRT67307 or 100 nM Ruxolitinib for 24, 48 and 72 hours.
For EZH2 inhibition experiments, H69 cells were treated with 5 μM GSK126 for 6 days. Drug was replenished every 3 days with both suspension and adherent cells carried each time. After the GSK126 treatment period, equal numbers of DMSO-treated and GSK126-treated cells were exposed to either H2O or 200 ng/mL IFN-γ for 24 hours before harvesting of RNA or conditioned media (CM).