Fig. 5: Sensitivity to TNF-induced septic shock in Dapk1−/− mice and DAPK1-deficiency selectively inhibits phosphorylation of RIPK1(S321), MK2, and p38 MAPK.
From: Tumor suppressor death-associated protein kinase 1 inhibits necroptosis by p38 MAPK activation
a, b Control and Dapk1−/− mice (n = 4 in each group) were injected with mouse TNF (1.0 μg/g body weight), and survival (a) and rectal body temperature (b) were determined at the indicated time points. P values (a) for Long-rank (Mantel-–Cox) test. Mean ± SD (b) are shown. *P < 0.05, **P < 0.01 (b) for two-way ANOVA followed by a Tukey’s multiple comparison test. (c) Normal TNF-triggered activation or NF-κB, ERK, JNK, and AKT in Dapk1−/− BMDMs. WT and Dapk1−/− BMDMs were treated with TNF (20 ng/ml), and cell lysates were prepared and levels of pIκBα, IκBα, pERK, ERK, pJNK, JNK, pAKT, and AKT were determined for the indicated time points. (d, f) Diminished TNF-induced phosphorylation of RIPK1(S321), MK2 and p38 MAPK in Dapk1−/− BMDMs. WT and Dapk1−/− BMDMs were treated with TNF (20 ng/ml), and levels of pIKK, IKK, pRIPK1(S321), RIPK1, p-p38, p38 (d), pMK2 and MK2 (f) were determined in cell lysates for the indicated time points. Data are representative of three independent experiments. (e) The extents of RIPK1(S321) and p38 MAPK activation from three independent experiments in (d) were quantitated using normalized intensity of pRIPK1(S321) and p-p38 in WT BMDMs at 15 min as 1. Mean ± SD are shown. *P < 0.05, ***P < 0.001 for two-way ANOVA followed by a Sidak’s multiple comparison test. (g) Inhibition of p38 MAPK or MK2 confers susceptibility to necroptosis in WT macrophages. WT BMDMs were treated with zVAD, AT-406, SB203589 (1 μM) and PF3644022 (2 μM), as indicated, and the extent of necroptosis determined. Values are mean ± SD of triplicates in a single experiment. **p < 0.01, ***p < 0.001 for unpaired t-test. Results have been repeated in two independent experiments.
