ADK prevents TNFα-induced cell death by reducing adenosine levels, which in turn licenses the methionine cycle and transmethylation reactions. (A) MEFs were treated with TNFα (10 ng/ml) for the specified durations (min). Then, the ADK subcellular localization was detected by immunofluorescence. (B) MEFs were transfected with lentivirus encoding ADK-L or ADK-S. Endogenous ADK was silenced with siRNA targeting 3′UTR. Then, the ADK subcellular localization was detected with immunofluorescence. (C and D) MEFs were transfected with lentivirus encoding ADK-L or ADK-S. Endogenous ADK was silenced with siRNA targeting 3′UTR. These cells were treated with TNFα (10 ng/ml) for the indicated time (C) or 12 h (D) in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (C). The levels of p-RIPK1(S166), CC8, CC3, and ADK were determined by immunoblotting (D). (E and F) Adenosine concentrations in primary hepatocytes or liver tissue of Adkf/f or Adkf/f;Alb-Cre mice were measured. (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 adenosine (1 mM). Cell death was measured by the SYTOX Green positivity assay (G). The levels of p-RIPK1(S166), CC8, and CC3 were determined with immunoblotting (H). (I and J) Primary hepatocytes from Ripk1WT/WT or Ripk1D138N/D138N mice were treated with TNFα (10 ng/ml) for the indicated time (I) or 12 h (J) in the presence or absence of 5-ITu (20 μM). Cell death was measured by the SYTOX Green positivity assay (I). The levels of p-RIPK1(S166), CC8, and CC3 were determined by immunoblotting (J). (K and L) Primary hepatocytes from Adkf/f or Adkf/f;Alb-Cre mice were treated with TNFα (10 ng/ml) for the indicated time (K) or 12 h (L) in the presence or absence of adenosine (1 mM). 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) Concentrations of SAM and SAH in primary hepatocytes or liver tissue of Adkf/f or Adkf/f;Alb-Cre mice were measured. (O and P) Primary hepatocytes from Adkf/f or Adkf/f;Alb-Cre mice were treated with TNFα (10 ng/ml) for the indicated time (O) or 12 h (P) in the presence or absence of SAM (100 μM). Cell death was measured by the SYTOX Green positivity assay (O). The levels of p-RIPK1(S166), CC8, CC3, and ADK were determined by immunoblotting (P). Data are represented as the mean ± SD (C, E–G, I, K, and M–O). Data are representative of n = 3 independent experiments (A–P). Statistical significance was determined using two-tailed unpaired Student’s t test (E, F, M, and N) or two-way ANOVA with post hoc Bonferroni’s test (C, G, I, K, and O). ***P < 0.001. Source data are available for this figure: SourceData F2.
Figure 2.

ADK prevents TNFα-induced cell death by reducing adenosine levels, which in turn licenses the methionine cycle and transmethylation reactions. (A) MEFs were treated with TNFα (10 ng/ml) for the specified durations (min). Then, the ADK subcellular localization was detected by immunofluorescence. (B) MEFs were transfected with lentivirus encoding ADK-L or ADK-S. Endogenous ADK was silenced with siRNA targeting 3′UTR. Then, the ADK subcellular localization was detected with immunofluorescence. (C and D) MEFs were transfected with lentivirus encoding ADK-L or ADK-S. Endogenous ADK was silenced with siRNA targeting 3′UTR. These cells were treated with TNFα (10 ng/ml) for the indicated time (C) or 12 h (D) in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (C). The levels of p-RIPK1(S166), CC8, CC3, and ADK were determined by immunoblotting (D). (E and F) Adenosine concentrations in primary hepatocytes or liver tissue of Adkf/f or Adkf/f;Alb-Cre mice were measured. (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 adenosine (1 mM). Cell death was measured by the SYTOX Green positivity assay (G). The levels of p-RIPK1(S166), CC8, and CC3 were determined with immunoblotting (H). (I and J) Primary hepatocytes from Ripk1WT/WT or Ripk1D138N/D138N mice were treated with TNFα (10 ng/ml) for the indicated time (I) or 12 h (J) in the presence or absence of 5-ITu (20 μM). Cell death was measured by the SYTOX Green positivity assay (I). The levels of p-RIPK1(S166), CC8, and CC3 were determined by immunoblotting (J). (K and L) Primary hepatocytes from Adkf/f or Adkf/f;Alb-Cre mice were treated with TNFα (10 ng/ml) for the indicated time (K) or 12 h (L) in the presence or absence of adenosine (1 mM). 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) Concentrations of SAM and SAH in primary hepatocytes or liver tissue of Adkf/f or Adkf/f;Alb-Cre mice were measured. (O and P) Primary hepatocytes from Adkf/f or Adkf/f;Alb-Cre mice were treated with TNFα (10 ng/ml) for the indicated time (O) or 12 h (P) in the presence or absence of SAM (100 μM). Cell death was measured by the SYTOX Green positivity assay (O). The levels of p-RIPK1(S166), CC8, CC3, and ADK were determined by immunoblotting (P). Data are represented as the mean ± SD (C, E–G, I, K, and M–O). Data are representative of n = 3 independent experiments (A–P). Statistical significance was determined using two-tailed unpaired Student’s t test (E, F, M, and N) or two-way ANOVA with post hoc Bonferroni’s test (C, G, I, K, and O). ***P < 0.001. Source data are available for this figure: SourceData F2.

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