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. 2021 Sep 2;24(9):103074. doi: 10.1016/j.isci.2021.103074

Figure 7.

Figure 7

Direct ERK1/2 inhibition efficiently impairs necroptosis driven by MLKL S345D overexpression in L929 cells

(A) MLKL-deficient L929 reconstituted with Dox-inducible phosphomimetic S345D mutant MLKL expression were used. These cells were pretreated for 1 h with the indicated compounds before Dox (red) or DMEM (blue) stimulation (grey curves). Cell death was measured as a function of time by SytoxGreen (SG+) positivity (kinetic experiment, left), 18 h after Dox stimulation (A, left histogram), and caspase-3 activity was assessed by DEVD-AMC cleavage efficiency (A, middle histogram) 18 h after Dox stimulation. Drugs-induced toxicity measurements (SG+ positivity) 18 h after DMEM stimulation (A, right histogram). The cell death 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; ns, not significant.

(B) Cells were pretreated or not for 1 h with the indicated compounds (10 μM) and subsequently stimulated with Dox. Cells were then lysed and immunoblotted as indicated on the left of each blot. Corresponding molecular weights are reported on the right of each blot. Both left and right parts came from the same blot; a cut was performed to remove non-relevant conditions.

(C) Biosensing experiment: Cells expressing the FRET biosensor EKAR4.0 were time lapse-imaged by FRET microscopy for 30 min before treatment (control & Dox) and 14.5 h after stimulation at the rate of 1 acquisition every 4 min. From left to right, the raw FRET/CFP ratio of individual cells is displayed by a FRET/CFP ratio kymographic representation (C, left), averaged raw FRET/CFP ratio (mean ± SEM) as a function of time (C, right).

(D) Corresponding quantitative analyses of ERK1/2 pulse dynamics are displayed by a binary kymographic representation of ERKON and ERKOFF states (D, left). As described in Figure 5, ERK1/2 activity is identified by red dots on one representative FRET/CFP ratio profile for each condition, plotted against time.

(E) Time-resolved quantitative analysis of EKAR4.0 pulse dynamics based on parameters identified in Figure 5 were calculated 1h after the start of imaging with a 2-h time interval, for each cell and for each condition (blue line: survival; red: necroptosis triggered by Dox-induced S345D MLKL expression). Temporal frame data analysis is presented as radar plots similar to Figure 5C. Dark spots and lines indicate the maximum for each parameter, while the center indicated the minimum. Min and max values are reported as follows: pulses width (6–15 min); pulses number (0.5–3); pulses amplitude (1.0–1.6); FRETON (4–16%). 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; ∗∗∗∗p< 0.0001; ns, not significant. Experimental and analysis protocols, as well as graphical representations, are identical to Figures 4 and 5.