Fig 2: RIPK1 knockdown facilitates the interaction between RIPK3 and TRADD in response to TNF stimulation. (A) The interaction between ectopically expressed TRADD and RIPK3. 293T cells were transfected with the indicated mammalian expression vectors. After 48 h, cells were collected and lyzed, and cell lysates were then subjected to immunoprecipitation (IP) with anti-FLAG or anti-HA antibody, followed by SDS-PAGE and immunoblot (IB) analysis with the indicated antibody. (B) The interaction between RIPK3 and TRADD significantly increased in RIPK1-knockdown L929 cells treated with TNF plus Z-VAD. RIPK1-knockdown and the negative control L929 cells were treated with TNF plus Z-VAD (T+Z) for the indicated length of time, and the interaction between RIPK3 and TRADD was determined by immunoprecipitation and western blot. Actin was used as a loading control. (C) TNF induces the interaction between RIPK3 and TRADD in RIPK1-knockdown L929 cells. RIPK1-knockdown and the negative control L929 cells were treated with TNF plus Z-VAD (T+Z) for the indicated length of time, and RIPK3-TRADD heterodimer were detected with a Duolink proximity ligation assay and confocal microscopy. All Duolink proximity ligation experiments were performed at least three times, and representative results are shown. The normalized signal per cell is shown as the mean ±SD of three separate experiments (n = 3). ∗∗P < 0.01. The RIPK1 knockdown efficiency was verified in Figure 1A.

Fig 3: TRADD is essential for RIPK3–MLKL signaling pathway activation triggered by TNF. (A) TRADD knockdown suppresses phosphorylation of RIPK3 and MLKL induced by TNF plus Z-VAD in the absence of RIPK1. L929 cells were infected with RIPK1 shRNA lentivirus with or without TRADD shRNA lentivirus for 48 h, and then treated with or without TNF plus Z-VAD (T+Z) for the indicated length of time. Western blot was used to detect the phosphorylation level of RIPK3 and MLKL (p-RIPK1 and p-MLKL) and the protein level of RIPK1 and TRADD. Actin was used as a loading control. (B) Restoration of TRADD expression recovers the activation of RIPK3-MLKL signaling pathway in the absence of RIPK1. RIPK1 and TRADD double-knockdown L929 cells were infected with pLV-Myc-TRADD and the negative control lentiviruses for 48 h, and then treated with or without TNF plus Z-VAD for 4 h. Western blotting was performed to evaluate the phosphorylation levels of RIPK3 and MLKL (p-RIPK3 and p-MLKL), and actin was used as a loading control. In addition, the expression level of TRADD, RIPK1, and Myc-tag was verified in Figure 2C. (C,E) TRADD knockdown suppresses TNF-induced RIPK3 and MLKL oligomerization in the absence of RIPK1. RIPK1-knockdown or RIPK1 and TRADD-double knockdown L929 cells were treated with or without TNF plus Z-VAD (T+Z), and lysed with cell lysis buffer containing or not containing β-ME. Western blotting was used to detect the monomer and oligomer of RIPK3 (Figure 3C) and MLKL (Figure 3E). Actin was used as a loading control. (D,F) TRADD is essential for the homo-interaction of RIPK3 or MLKL in response to TNF stimulation. RIPK1 knockdown or RIPK1 and TRADD double-knockdown L929 cells were treated with TNF plus Z-VAD (T+Z) for the indicated length of time, and the homodimers of RIPK3 or MLKL were detected with a Duolink proximity ligation assay and confocal microscopy. All Duolink proximity ligation experiments were performed at least three times, and representative results are shown. The normalized signal per cell is shown as the mean ±SD of three separate experiments (n = 3). ∗∗P < 0.01.

Fig 4: Analysis of necroptotic signaling in the left and right ventricle of rat hearts. (A) representative immunoblots and total protein staining; (B–F) Immunoblot quantification of RIP1, RIP3, pSer229-RIP3, ratio pSer229-RIP3/RIP3 and MLKL. Sham—sham-operated group; HF—group with post-myocardial infarction heart failure; LV—left ventricle; RV—right ventricle. Data are presented as mean ± SEM; n = 6–9 per group; 2WA—2-way ANOVA; “HF” factor—presence of heart failure; “V” factor—heart ventricle; “Interaction” of 2 factors. * p < 0.05 vs. Sham.

Fig 5: Analysis of activation of canonical necroptotic pathway in left ventricles lysates. Immunoblot quantification of RIP1 (a), pThr231/Ser232-RIP3/RIP3 ratio (b) and MLKL (c); representative immunoblots and total protein staining (d). Data are presented as mean ± SEM.