Adenosine is indispensable for ADK in regulating TNFα-induced cell death. (A) Primary hepatocytes were isolated from Adkf/f or Adkf/f;Alb-Cre mice. Intracellular ATP, ADP, and AMP levels were measured. (B) MEFs were transfected with siNC or siAdk. Intracellular adenosine, ATP, ADP, and AMP levels were measured. (C) MEFs were treated with vehicle or adenosine (1 mM). After 1 h, intracellular adenosine, ATP, ADP, and AMP levels were measured. (D) Primary hepatocytes were isolated from WT mice and were treated with vehicle or adenosine (1 mM). After 1 h, intracellular adenosine level was measured. (E and F) Primary hepatocytes were treated with TNFα (10 ng/ml) for the indicated time (E) or 12 h (F) in the presence or absence of adenosine (1 mM) or Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (E). The levels of p-RIPK1(S166), CC8, and CC3 were determined by immunoblotting (F). (G and H) Primary hepatocytes were treated with TNFα (10 ng/ml) for the indicated time (G) or 12 h (H) in the presence or absence of 5-ITu (20 μM) or 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 by immunoblotting (H). (I and J) MEFs transfected with siNC or siAdora1/2a/2b/3 were treated with TNFα (10 ng/ml) for the indicated time (I) or 12 h (J) in the presence or absence of adenosine (1 mM). Cell death was measured by the SYTOX Green positivity assay (I). The levels of p-RIPK1(S166), CC8, CC3, and adenosine receptors were determined by immunoblotting (J). (K) Schematic diagram of ADK in the regulation of the methionine cycle. (L) MEFs were treated with vehicle or adenosine (1 mM). After 1 h, intracellular SAM and SAH were measured and their ratio was calculated. (M) MEFs were transfected with siNC or siAdk. Intracellular SAM and SAH were measured, and their ratio was calculated. (N and O) MEFs transfected with siNC or siAdk were treated with TNFα (10 ng/ml) for the indicated time (N) or 12 h (O) in the presence or absence of SAM (100 μM). Cell death was measured by the SYTOX Green positivity assay (N). The levels of p-RIPK1(S166), CC8, CC3, and ADK were determined by immunoblotting (O). (P and Q) MEFs transfected with siNC or siAdk were treated with TNFα (10 ng/ml) for the indicated time (P) or 12 h (Q) in the presence or absence of DZNep (10 μM). Cell death was measured by the SYTOX Green positivity assay (P). The levels of p-RIPK1(S166), CC8, CC3, and ADK were determined by immunoblotting (Q). Data are represented as mean ± SD (A–E, G, I, L, M, N, and P). Data are representative of n = 3 independent experiments (A–Q). Statistical significance was determined using unpaired two-tailed Student’s t test (A–D, L, and M) or two-way ANOVA with post hoc Bonferroni’s test (E, G, I, N, and P). *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData FS2.
Figure S2.

Adenosine is indispensable for ADK in regulating TNFα-induced cell death. (A) Primary hepatocytes were isolated from Adkf/f or Adkf/f;Alb-Cre mice. Intracellular ATP, ADP, and AMP levels were measured. (B) MEFs were transfected with siNC or siAdk. Intracellular adenosine, ATP, ADP, and AMP levels were measured. (C) MEFs were treated with vehicle or adenosine (1 mM). After 1 h, intracellular adenosine, ATP, ADP, and AMP levels were measured. (D) Primary hepatocytes were isolated from WT mice and were treated with vehicle or adenosine (1 mM). After 1 h, intracellular adenosine level was measured. (E and F) Primary hepatocytes were treated with TNFα (10 ng/ml) for the indicated time (E) or 12 h (F) in the presence or absence of adenosine (1 mM) or Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (E). The levels of p-RIPK1(S166), CC8, and CC3 were determined by immunoblotting (F). (G and H) Primary hepatocytes were treated with TNFα (10 ng/ml) for the indicated time (G) or 12 h (H) in the presence or absence of 5-ITu (20 μM) or 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 by immunoblotting (H). (I and J) MEFs transfected with siNC or siAdora1/2a/2b/3 were treated with TNFα (10 ng/ml) for the indicated time (I) or 12 h (J) in the presence or absence of adenosine (1 mM). Cell death was measured by the SYTOX Green positivity assay (I). The levels of p-RIPK1(S166), CC8, CC3, and adenosine receptors were determined by immunoblotting (J). (K) Schematic diagram of ADK in the regulation of the methionine cycle. (L) MEFs were treated with vehicle or adenosine (1 mM). After 1 h, intracellular SAM and SAH were measured and their ratio was calculated. (M) MEFs were transfected with siNC or siAdk. Intracellular SAM and SAH were measured, and their ratio was calculated. (N and O) MEFs transfected with siNC or siAdk were treated with TNFα (10 ng/ml) for the indicated time (N) or 12 h (O) in the presence or absence of SAM (100 μM). Cell death was measured by the SYTOX Green positivity assay (N). The levels of p-RIPK1(S166), CC8, CC3, and ADK were determined by immunoblotting (O). (P and Q) MEFs transfected with siNC or siAdk were treated with TNFα (10 ng/ml) for the indicated time (P) or 12 h (Q) in the presence or absence of DZNep (10 μM). Cell death was measured by the SYTOX Green positivity assay (P). The levels of p-RIPK1(S166), CC8, CC3, and ADK were determined by immunoblotting (Q). Data are represented as mean ± SD (A–E, G, I, L, M, N, and P). Data are representative of n = 3 independent experiments (A–Q). Statistical significance was determined using unpaired two-tailed Student’s t test (A–D, L, and M) or two-way ANOVA with post hoc Bonferroni’s test (E, G, I, N, and P). *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData FS2.

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