RIPK1 R606 symmetric dimethylation is catalyzed by arginine methyltransferase PRMT5. (A and B) Primary hepatocytes were transfected with siNC or siRNAs targeting PRMTs. The cells were treated with 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 p-RIPK1(S166), CC8, and CC3 were determined by immunoblotting (B). (C) HEK293T cells were cotransfected with expression vectors for Flag-RIPK1, as well as PRMT5, PRMT7, or PRMT9. Flag-RIPK1 was immunoprecipitated using anti-Flag antibody, and R606me2s level was determined by immunoblotting. (D) HEK293T cells were transfected with expression vectors for Flag-RIPK1. Endogenous PRMT5 was deleted with the CRISPR/Cas9 system. Flag-RIPK1 was immunoprecipitated using anti-Flag antibody, and R606me2s level was determined by immunoblotting. (E) HEK293T cells were cotransfected with expression vectors for HA-RIPK1 and Flag-PRMT5. The interaction between HA-RIPK1 and Flag-PRMT5 was analyzed with immunoprecipitation and immunoblotting. (F) MEFs transfected with siNC or siPrmt5 were treated with TNFα (10 ng/ml) for 15 min. The levels of RIPK1 R606me2s and PRMT5 were determined by immunoblotting. (G) WT or R606K mutant RIPK1 was purified from PRMT5−/− HEK293T cells and was subjected to in vitro methylation assays for 1 h in the presence or absence of WT or G367A/R368A mutant PRMT5, EPZ015666, MEP50, SAM. The levels of RIPK1 R606me2s, PRMT5, and RIPK1 were determined by immunoblotting. The generation of SAH was analyzed via the MTase-Glo methyltransferase assay kit. (H and I) Primary hepatocytes from Adkf/f or Adkf/f;Alb-Cre mice were treated with TNFα (10 ng/ml) for the indicated time (H) or 12 h (I) with or without siPrmt5 in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (H). The levels of p-RIPK1(S166), CC8, CC3, ADK, and PRMT5 were determined by immunoblotting (I). (J and K) Primary hepatocytes were treated with TNFα (10 ng/ml) for the indicated time (J) or 12 h (K) with or without siPrmt5 in the presence or absence of adenosine (1 mM) or Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (J). The levels of p-RIPK1(S166), CC8, CC3, and PRMT5 were determined by immunoblotting (K). Data are represented as the mean ± SD (A, G, H, and J). Data are representative of n = 3 independent experiments (A–K). Statistical significance was determined using two-way ANOVA with post hoc Bonferroni’s test (A, H, and J) or one-way ANOVA with post hoc Dunnett’s test (G). **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData F6.
Figure 6.

RIPK1 R606 symmetric dimethylation is catalyzed by arginine methyltransferase PRMT5. (A and B) Primary hepatocytes were transfected with siNC or siRNAs targeting PRMTs. The cells were treated with 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 p-RIPK1(S166), CC8, and CC3 were determined by immunoblotting (B). (C) HEK293T cells were cotransfected with expression vectors for Flag-RIPK1, as well as PRMT5, PRMT7, or PRMT9. Flag-RIPK1 was immunoprecipitated using anti-Flag antibody, and R606me2s level was determined by immunoblotting. (D) HEK293T cells were transfected with expression vectors for Flag-RIPK1. Endogenous PRMT5 was deleted with the CRISPR/Cas9 system. Flag-RIPK1 was immunoprecipitated using anti-Flag antibody, and R606me2s level was determined by immunoblotting. (E) HEK293T cells were cotransfected with expression vectors for HA-RIPK1 and Flag-PRMT5. The interaction between HA-RIPK1 and Flag-PRMT5 was analyzed with immunoprecipitation and immunoblotting. (F) MEFs transfected with siNC or siPrmt5 were treated with TNFα (10 ng/ml) for 15 min. The levels of RIPK1 R606me2s and PRMT5 were determined by immunoblotting. (G) WT or R606K mutant RIPK1 was purified from PRMT5−/− HEK293T cells and was subjected to in vitro methylation assays for 1 h in the presence or absence of WT or G367A/R368A mutant PRMT5, EPZ015666, MEP50, SAM. The levels of RIPK1 R606me2s, PRMT5, and RIPK1 were determined by immunoblotting. The generation of SAH was analyzed via the MTase-Glo methyltransferase assay kit. (H and I) Primary hepatocytes from Adkf/f or Adkf/f;Alb-Cre mice were treated with TNFα (10 ng/ml) for the indicated time (H) or 12 h (I) with or without siPrmt5 in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (H). The levels of p-RIPK1(S166), CC8, CC3, ADK, and PRMT5 were determined by immunoblotting (I). (J and K) Primary hepatocytes were treated with TNFα (10 ng/ml) for the indicated time (J) or 12 h (K) with or without siPrmt5 in the presence or absence of adenosine (1 mM) or Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (J). The levels of p-RIPK1(S166), CC8, CC3, and PRMT5 were determined by immunoblotting (K). Data are represented as the mean ± SD (A, G, H, and J). Data are representative of n = 3 independent experiments (A–K). Statistical significance was determined using two-way ANOVA with post hoc Bonferroni’s test (A, H, and J) or one-way ANOVA with post hoc Dunnett’s test (G). **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData F6.

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