Identification of ADK as a suppressor of TNFα-induced RIPK1-driven cell death. (A and B) Primary hepatocytes from Adkf/f or Adkf/f;Alb-Cre mice were treated with CHX (1 μM)/TNFα (10 ng/ml) (T/C) for the indicated time (A) or 12 h (B). Cell death was measured by the SYTOX Green positivity assay (A). The levels of cleaved caspase-8 (CC8), cleaved caspase-3 (CC3), and ADK were determined by immunoblotting (B). Scale bar: 50 μm. (C and D) Primary hepatocytes from Adkf/f or Adkf/f;Alb-Cre mice were treated with TNFα (10 ng/ml) for the indicated time (C) or 12 h (D). Cell death was measured by the SYTOX Green positivity assay (C). The levels of CC8, CC3, and ADK were determined by immunoblotting (D). (E and F) Primary hepatocytes from Adkf/f or Adkf/f;Alb-Cre mice were treated with CHX (1 μM)/TNFα (10 ng/ml) for the indicated time (E) or 12 h (F) in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (E). The levels of p-RIPK1(S166), CC8, CC3, and ADK were determined by immunoblotting (F). (G and H) Primary hepatocytes from Adkf/f or Adkf/f;Alb-Cre mice were treated with TNFα (10 ng/ml) for the indicated time (G) or 12 h (H) in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (G). The levels of p-RIPK1(S166), CC8, CC3, and ADK were determined by immunoblotting (H). (I and J) Primary hepatocytes from Adkf/f, Adkf/f;Alb-Cre, and Adkf/f;Alb-Cre;Ripk1D138N/D138N mice were treated with CHX (1 μM)/TNFα (10 ng/ml) for the indicated time (I) or 12 h (J). Cell death was measured by the SYTOX Green positivity assay (I). The levels of p-RIPK1(S166), CC8, CC3, and ADK were determined by immunoblotting (J). (K and L) Primary hepatocytes from Adkf/f, Adkf/f;Alb-Cre, and Adkf/f;Alb-Cre;Ripk1D138N/D138N mice were treated with TNFα (10 ng/ml) for the indicated time (K) or 12 h (L). Cell death was measured by the SYTOX Green positivity assay (K). The levels of p-RIPK1(S166), CC8, CC3, and ADK were determined by immunoblotting (L). (M) MEFs transfected with siRNAs of negative control (siNC) or siRNAs targeting ADK (siAdk) were stimulated by Flag-TNFα (100 ng/ml) for the indicated time. The TNFR1 signaling complex was immunoprecipitated using an anti-Flag antibody. The complexes were analyzed by immunoblotting using anti-p-S166 RIPK1 antibody and other antibodies as indicated. (N) MEFs transfected with siNC or siAdk were preincubated with zVAD.fmk (10 μM) in the presence or absence of Nec-1s (10 μM) for 0.5 h and then stimulated with 10 ng/ml TNFα for 12 h. The complex II was isolated by FADD immunoprecipitation, and RIPK1 binding was revealed by immunoblotting. (O) MEFs transfected with siNC or siAdk were pretreated with CHX (C, 2 μg/ml) and zVAD.fmk (Z, 10 μM) for 0.5 h followed by 10 ng/ml TNFα (T) for the indicated time. The necrosome was isolated by immunoprecipitation of RIPK3, and RIPK1 binding was revealed by immunoblotting. Data are represented as the mean ± SD (A, C, E, G, I, and K). Data are representative of n = 3 independent experiments (A–O). Statistical significance was determined using two-way ANOVA with post hoc Bonferroni’s test (A, C, E, G, I, and K). ***P < 0.001. Source data are available for this figure: SourceData F1.
Figure 1.

Identification of ADK as a suppressor of TNFα-induced RIPK1-driven cell death. (A and B) Primary hepatocytes from Adkf/f or Adkf/f;Alb-Cre mice were treated with CHX (1 μM)/TNFα (10 ng/ml) (T/C) for the indicated time (A) or 12 h (B). Cell death was measured by the SYTOX Green positivity assay (A). The levels of cleaved caspase-8 (CC8), cleaved caspase-3 (CC3), and ADK were determined by immunoblotting (B). Scale bar: 50 μm. (C and D) Primary hepatocytes from Adkf/f or Adkf/f;Alb-Cre mice were treated with TNFα (10 ng/ml) for the indicated time (C) or 12 h (D). Cell death was measured by the SYTOX Green positivity assay (C). The levels of CC8, CC3, and ADK were determined by immunoblotting (D). (E and F) Primary hepatocytes from Adkf/f or Adkf/f;Alb-Cre mice were treated with CHX (1 μM)/TNFα (10 ng/ml) for the indicated time (E) or 12 h (F) in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (E). The levels of p-RIPK1(S166), CC8, CC3, and ADK were determined by immunoblotting (F). (G and H) Primary hepatocytes from Adkf/f or Adkf/f;Alb-Cre mice were treated with TNFα (10 ng/ml) for the indicated time (G) or 12 h (H) in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (G). The levels of p-RIPK1(S166), CC8, CC3, and ADK were determined by immunoblotting (H). (I and J) Primary hepatocytes from Adkf/f, Adkf/f;Alb-Cre, and Adkf/f;Alb-Cre;Ripk1D138N/D138N mice were treated with CHX (1 μM)/TNFα (10 ng/ml) for the indicated time (I) or 12 h (J). Cell death was measured by the SYTOX Green positivity assay (I). The levels of p-RIPK1(S166), CC8, CC3, and ADK were determined by immunoblotting (J). (K and L) Primary hepatocytes from Adkf/f, Adkf/f;Alb-Cre, and Adkf/f;Alb-Cre;Ripk1D138N/D138N mice were treated with TNFα (10 ng/ml) for the indicated time (K) or 12 h (L). Cell death was measured by the SYTOX Green positivity assay (K). The levels of p-RIPK1(S166), CC8, CC3, and ADK were determined by immunoblotting (L). (M) MEFs transfected with siRNAs of negative control (siNC) or siRNAs targeting ADK (siAdk) were stimulated by Flag-TNFα (100 ng/ml) for the indicated time. The TNFR1 signaling complex was immunoprecipitated using an anti-Flag antibody. The complexes were analyzed by immunoblotting using anti-p-S166 RIPK1 antibody and other antibodies as indicated. (N) MEFs transfected with siNC or siAdk were preincubated with zVAD.fmk (10 μM) in the presence or absence of Nec-1s (10 μM) for 0.5 h and then stimulated with 10 ng/ml TNFα for 12 h. The complex II was isolated by FADD immunoprecipitation, and RIPK1 binding was revealed by immunoblotting. (O) MEFs transfected with siNC or siAdk were pretreated with CHX (C, 2 μg/ml) and zVAD.fmk (Z, 10 μM) for 0.5 h followed by 10 ng/ml TNFα (T) for the indicated time. The necrosome was isolated by immunoprecipitation of RIPK3, and RIPK1 binding was revealed by immunoblotting. Data are represented as the mean ± SD (A, C, E, G, I, and K). Data are representative of n = 3 independent experiments (A–O). Statistical significance was determined using two-way ANOVA with post hoc Bonferroni’s test (A, C, E, G, I, and K). ***P < 0.001. Source data are available for this figure: SourceData F1.

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