Ulk1/2+/+ and Ulk1/2−/− MEFs were seeded in quadruplicate in 96-well plates overnight and then treated with the indicated doses of mouse IFN-γ for 16 hours. Cells were subsequently challenged with EMCV, and EMCV-induced CPEs were determined 24 hours later. Briefly, medium was aspirated, and cells were fixed in 95% ethanol for 30 min and then stained with 0.1% crystal violet in 2% ethanol for a further 30 min. Cells were then destained in 0.5 M NaCl in 50% ethanol for 1 hour, and then, absorbance readings at 570 nm were taken to determine the extent of uninfected, viable cells. For those assays where the ULK1 pharmacological inhibitor MRT68921 or the ERK5 inhibitor XMD8-92 was included, human fibrosarcoma 2fTGH cells were seeded in quadruplicate in 96-well plates and then either treated with human IFN-γ for 16 hours or pretreated for 2 hours with MRT68921 (1 μM) or with XMD8-92 (5 μM) and then, the medium was removed, and the cells were treated with MRT68921 or XMD8-92 and human IFN-γ, as indicated, for a further 16 hours. Cells were subsequently challenged with EMCV, and EMCV-induced CPEs were determined 24 hours later, as described above.

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