Figure 1.

Effect of direct ERK1/2 inhibition in TNF-induced necroptosis and hFasL-induced apoptosis

(A) L929 wildtype cells were pretreated for 1 h with the indicated compounds, all targeting the MAPK/ERK signaling pathway (Figure S1A) or RIPK1 activity (grey curves) before TNF (red) or control (blue) treatment.

(B and C) SCH772984 dose-response in TNF-induced necroptosis, in the presence (C) or the absence (B) of the pan-caspase inhibitor zVAD-fmk (zVAD).

(D) Sensitizing effect of SCH772984 in hFas induced apoptosis. For each panel, cell death was measured as (i) function of time by SytoxGreen (SG⁺) positivity (A–D, left graph), and (ii) plotted at 10 h after treatment (A–D, middle graph). Caspase-3 activity was assessed by DEVD-AMC cleavage efficiency (A–D, right graph). Data are presented as mean ± SEM of at least two independent experiments. Statistical significance was determined using two-way ANOVA followed by Tukey’s post hoc test. Significance between samples is indicated as follows: ^∗p < 0.05; ^∗∗p< 0.01; ^∗∗∗p< 0.001.

(E) L929 cells were pre-treated or not with the indicated compounds or DMSO for 30 min before TNF treatment. After 6 h, cells were then lysed and immunoblotted as indicated on the left of each blot.