Fig. 4

ERK activity is essential for cell death after IFNγ treatment. (A) M238 cells were treated in triplicate wells as labeled on the x-axis. Cells were stained on day 5 and imaged to enumerate total DNA-containing objects (left panel) and those with caspase 3/7 activity (right panel). Caspase activity counts were normalized by the total DNA-containing object counts, and means were plotted in the right panel. Error bars indicate SEM. Results are representative of 3 independent experiments. Statistically significant differences in cell death compared to the IFNγ-treated sample are indicated by an asterisk: * P < 0.05, ** P < 0.01. (B) M238 cells were plated with the indicated treatments (top) in triplicate wells per condition, along with the Cytotox Red dye, which stains dead or dying cells with compromised membrane integrity. Plots show changes in confluence (left panel) and dead cell count (right panel) over time. The area under the curve (AUC) was calculated for each curve and converted into a heatmap, as shown below in the respective plot. (C) Live imaging experiments, similar to panel B, were set up using 23 melanoma lines. AUC values were calculated as in B, and the resulting heatmaps are shown for cell growth (left) and cell death (right) curves for each line. The annotation to the left shows the molecular subtype of each tested melanoma line. The vertical bracket indicates 17 melanoma lines in which IFNγ-mediated cell death is rescued by ulixertinib treatment. Results are representative of 2 independent experiments. Heatmap labels for M230 and M238 lines, compared in an RNA-seq experiment in Fig. 5, are indicated with an underline. (D) M238 cells were treated with different IFNγ and PMA doses as indicated on the x and y axis, respectively, with four replicates per condition. The red color indicates areas of synergy between the two treatments with respect to growth inhibition. Area of maximum synergy is indicated with a box. Data is representative of 3 independent experiments. See also Fig. S8