ADK inhibits RIPK1 kinase–driven cell death induced by TNFα. (A and B) MEFs transfected with siNC or siAdk were treated with CHX (1 μM)/TNFα (10 ng/ml) for the indicated time (A) or 12 h (B). Cell death was measured by the SYTOX Green positivity assay (A). The levels of CC8, CC3, and ADK were determined by immunoblotting (B). (C and D) MEFs transfected with siNC or siAdk 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) MEFs transfected with siNC or siAdk were treated with TNFα (10 ng/ml) for the specified durations (min). Activation of MAPK or NF-κB pathways was analyzed with immunoblotting. (F) MEFs transfected with siNC or siAdk were treated with TNFα (10 ng/ml) for the specified durations. Then, the p65 nucleus translocation level of MEFs was detected by immunofluorescence. Scale bar: 20 μm. (G and H) MEFs transfected with siNC or siAdk were treated with CHX (1 μM)/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) MEFs transfected with siNC or siAdk were treated with TNFα (10 ng/ml) for the indicated time (I) or 12 h (J) in the presence or absence of Nec-1s (10 μM). 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) MEFs transfected with siNC or siAdk were treated with 5z7 (500 nM)/TNFα (10 ng/ml) for the indicated time (K) or 12 h (L) in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (K). The levels of p-RIPK1(S166), CC8, and CC3 were determined by immunoblotting (L). (M and N) MEFs transfected with siNC or siAdk were treated with CHX (2 μg/ml) and zVAD.fmk (Z, 10 μM) for 0.5 h followed by 10 ng/ml TNFα for the indicated time. Cell death was measured by the SYTOX Green positivity assay (M). The levels of p-S166 RIPK1, p-T231/S232 RIPK3, and p-S345 MLKL were determined by immunoblotting (N). Data are represented as the mean ± SD (A, C, F, G, I, K, and M). Data are representative of n = 3 independent experiments (A–N). Statistical significance was determined using two-way ANOVA with post hoc Bonferroni’s test (A, C, F, G, I, K, and M). **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData FS1.
Figure S1.

ADK inhibits RIPK1 kinase–driven cell death induced by TNFα. (A and B) MEFs transfected with siNC or siAdk were treated with CHX (1 μM)/TNFα (10 ng/ml) for the indicated time (A) or 12 h (B). Cell death was measured by the SYTOX Green positivity assay (A). The levels of CC8, CC3, and ADK were determined by immunoblotting (B). (C and D) MEFs transfected with siNC or siAdk 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) MEFs transfected with siNC or siAdk were treated with TNFα (10 ng/ml) for the specified durations (min). Activation of MAPK or NF-κB pathways was analyzed with immunoblotting. (F) MEFs transfected with siNC or siAdk were treated with TNFα (10 ng/ml) for the specified durations. Then, the p65 nucleus translocation level of MEFs was detected by immunofluorescence. Scale bar: 20 μm. (G and H) MEFs transfected with siNC or siAdk were treated with CHX (1 μM)/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) MEFs transfected with siNC or siAdk were treated with TNFα (10 ng/ml) for the indicated time (I) or 12 h (J) in the presence or absence of Nec-1s (10 μM). 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) MEFs transfected with siNC or siAdk were treated with 5z7 (500 nM)/TNFα (10 ng/ml) for the indicated time (K) or 12 h (L) in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (K). The levels of p-RIPK1(S166), CC8, and CC3 were determined by immunoblotting (L). (M and N) MEFs transfected with siNC or siAdk were treated with CHX (2 μg/ml) and zVAD.fmk (Z, 10 μM) for 0.5 h followed by 10 ng/ml TNFα for the indicated time. Cell death was measured by the SYTOX Green positivity assay (M). The levels of p-S166 RIPK1, p-T231/S232 RIPK3, and p-S345 MLKL were determined by immunoblotting (N). Data are represented as the mean ± SD (A, C, F, G, I, K, and M). Data are representative of n = 3 independent experiments (A–N). Statistical significance was determined using two-way ANOVA with post hoc Bonferroni’s test (A, C, F, G, I, K, and M). **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData FS1.

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