Fig 1: The RIPK1/RIPK3-complex is required for YQJPF to inhibit hepatocyte necroptosis in vitro. Human LO2 cells were treated with LPS + D-Gal for 4 h and further with YQJPF and atractylone. (A,B) Immunofluorescence staining of RIPK1 and RIPK3 in LO2 cells. Scale bar, 50 µm. (C) Western blot analysis and quantitative assessment of RIPK1, MLKL and RIPK3. (D,E) Expression levels of RIPK1 and RIPK3 in LO2 were detected by ELISA. (F) RIPK1 was immunoprecipitated with its antibody and resulted in co-immunoprecipitation of RIPK3. Immunoprecipitation of RIPK3 with its antibody caused co-immunoprecipitation of RIPK1 in LO2 cells. Data are expressed as mean ± SD (n = 3). *vs. control group, #vs. LPS + D-Gal group; *p < 0.05, **p < 0.01; # p < 0.05, ## p < 0.01.
Fig 2: IL-17B is highly expressed in OGD cells and promotes the activation of the RIP3/MLKL pathway. IL-17B is highly expressed in OGD cells and promotes the activation of the RIP3/MLKL pathway. (a) Western blot for the detection of IL-17B protein in the OGD cells using β-actin as the internal reference. The line chart showed the expression trend of IL-17B protein at different OGD time. (b) Western blot for the detection of RIP3, p-RIP3, MLKL, and p-MLKL proteins in each cell group, using β-actin as the internal reference. The ImageJ image software analysis was used to test and analyze the expression ratios. (c) The bar chart shows the ratio of protein gray value to total protein gray value. ANOVA was used for statistical analysis. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
Fig 3: RIPK3 promotes TNFa-induced INS-1 cell death when caspases are inhibited. A) NIT-1 CTL and NIT-1 RIPK3? cell death was quantified 48 h post treatment (n = 3). Cell culture treatment conditions are indicated by color (black: vehicle, blue: 40 ng/ml TNFa, green: 40 ng/ml TNFa + 50 µM zVAD) and ß-cell lines are indicated by shapes (NIT-1 CTL: circles, NIT-1 RIPK3?: diamonds, INS-1: squares, INS-1 Empty: triangles, INS-1 mRIPK3: hexagons). B) NIT-1 CTL and RIPK3? cell caspase 3/7 activity was quantified 24 h post treatment and expressed relative to vehicle treated NIT-1 CTL cells (n = 4). C) INS-1 cell death was quantified 24 h post treatment relative to t = 0 (n = 4–9). Cell culture treatment conditions are indicated by color (black: vehicle, white: 5 µM GSK’872, blue: 40 ng/ml TNFa, light blue: TNFa+GSK’872, green: TNFa + 50 µM zVAD, light green: TNFa+zVAD + GSK’872). D) INS-1 cell caspase 3/7 activity was quantified 24 h post treatment and expressed relative to vehicle treated cells (n = 3). E)Ripk3 RNA expression (n = 4) and F) RIPK3 protein expression were quantified in control (pcDNA3-Empty: triangles) and Ripk3 overexpressing (pcDNA3-mRipk3: hexagons) INS-1 cells. G) INS-1 pcDNA3-Empty and pcDNA3-mRipk3 cell death was quantified 24 h post TNFa or TNFa+zVAD treatment (n = 4). H) Immunoblot analysis of proteins immunoprecipitated with anti-RIPK3 following 24 h TNFa+zVAD treatment (n = 3). I) Immunoblot analysis of proteins immunoprecipitated with anti-MLKL following 24 h TNFa+zVAD treatment (n = 3). Data are presented as mean ± SEM and were analyzed by one-way ANOVA followed by Sidák post-test and multiple comparisons correction. *p < 0.05; **p < 0.01; ***p < 0.001; ns, p > 0.05 as indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig 4: TRIM25 promotes the proteasomal degradation of RIP3.a Immunoblotting analysis of RIP1, RIP3, and MLKL in WT and TRIM25 KO HT-29 cells. b FLAG-tagged RIP3 was transfected into HeLa cells, then followed by the treatment with siRNAs of TRIM25. c FLAG-tagged RIP3 was co-transfected with or without Myc-tagged TRIM25 into HeLa cells, followed by the treatment by adding 10 uM MG132. d FLAG-tagged RIP3 was co-transfected with Myc-tagged TRIM25 or its mutant TRIM25CS into HeLa cells, followed by the treatment with adding 10 uM MG132. e TRIM25 or TRIM25CS were co-transfected with RIP3 in a dose-dependent manner in HeLa cells. f FLAG-tagged RIP3 was co-transfected with or without Myc-tagged TRIM25 into HeLa cells, followed by the treatment with MG132 (10 uM) or chloroquine (10 uM) for 4 h.
Fig 5: Quantification of nuclear and intercalated disc p-MLKL levels. (A) The number of phosphorylated MLKL (p-MLKL)-positive cardiomyocyte nuclei and the number of total cardiomyocyte nuclei in myocardial sections were counted, and nuclear p-MLKL level was expressed as the percentage of the number of p-MLKL-positive cardiomyocyte nuclei to the number of total cardiomyocyte nuclei. Using the median value, that is, 25%, patients were classified into a high nuclear p-MLKL level group (H-nucMLKL) and a low nuclear p-MLKL level group (L-nucMLKL). (B) p-MLKL expression levels in the intercalated discs of cardiomyocytes were analysed in a field (200 × 200 µm) in which cardiomyocytes were sectioned longitudinally. Intercalated disc areas stained with anti-p-MLKL antibodies were selected and quantified using BZ-X analyzer software, and the percentage of the positive area to the total area of cardiomyocytes was calculated. Using the median value, that is, 0.38%, patients were classified into a high intercalated disc p-MLKL level group (H-idMLKL) and a low intercalated disc p-MLKL level group (L-idMLKL).
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